Figure 7A ) Binding of NDP52 to ubiquitin tetramers of different linkage types . Beads coated with the indicated GST - tagged proteins were incubated with pooled M1 - , K63 - , and K48 - linked tetra - ubiquitin chains . Anti - ubiquitin Western blot of input and proteins bound to beads . B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates . B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates . B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Binding of NDP52 to ubiquitin tetramers of different linkage types. Beads coated with the indicated GST-tagged proteins were incubated with pooled M1-, K63-, and K48-linked tetra-ubiquitin chains. Anti-ubiquitin Western blot of input and proteins bound to beads.B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


Figure 2 ( B , C ) Representative immunoblots ( B ) and quantification of Tau degradation ( C ) from 14 DIV primary neurons transduced with EGFP or shTSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shTSG101 - expressing neurons exhibit markedly decreased Tau degradation compared to EGFP - expressing controls ( n = 18 - 19 per condition , unpaired student ' s t - test , * p = 0 . 0108 ) ( D , E ) Representative immunoblots ( D ) and quantification of Tau degradation ( E ) from 14 DIV neurons transduced with mCh or mCh - TSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . Overexpression of TSG101 increases Tau degradation ( n = 17 / condition , unpaired student ' s t - test , * p = 0 . 0115 ) ( F , G ) Representative immunoblots ( F ) and quantification of Tau degradation ( G ) from 14 DIV neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shRab35 - expressing neurons exhibit markedly decreased Tau degradation compared to mCh - expressing controls ( n = 23 - 26 per condition , unpaired student ' s t - test , * * * p = 0 . 0008 ) ( H , I ) Representative immunoblots ( H ) and quantification of Tau degradation ( I ) from 14 DIV neurons transduced with mCh or mCh - Rab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau or tubulin . Rab35 overexpression increases Tau degradation ( n = 18 per condition , unpaired student ' s t - test , * p = 0 . 0126 ) ( J ) Flow cytometry distribution of N2a cells cotransfected with either HA vector ( black ) or HA - Rab35 ( green ) and medium fluoresence timer - tagged wild - type Tau ( FT - Tau ) ; blue fluorescence ( y axis ) indicates " younger " Tau protein while red fluorescence ( x axis ) indicates " older " Tau ( K ) The ratio of cells expressing red to blue ( older : younger ) FT - Tau is reduced in cells overexpressing Rab35 , indicating faster Tau turnover ( n = 9 / condition , unpaired student ' s t - test , * * * * p < 0 . 0001 )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108)  (D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115)  (F, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008) (H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126) (J) Flow cytometry distribution of N2a cells cotransfected with either HA vector (black) or HA-Rab35 (green) and medium fluoresence timer-tagged wild-type Tau (FT-Tau); blue fluorescence (y axis) indicates "younger" Tau protein while red fluorescence (x axis) indicates "older" Tau (K) The ratio of cells expressing red to blue (older:younger) FT-Tau is reduced in cells overexpressing Rab35, indicating faster Tau turnover (n=9/condition, unpaired student's t-test , ****p<0.0001)


Figure 3 ( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) from 14 DIV primary neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for pSer396 / 404 - Tau ( PHF1 ) , pSer262 - Tau , or pSer202 - Tau ( CP13 ) and tubulin . shRab35 - expressing neurons exhibit markedly decreased pSer262 - and p396 / 404 - Tau degradation compared to mCh - expressing controls , while pSer202 - Tau degradation is unaffected ( n = 4 per condition for pSer396 / 404 - Tau and pSer202 - Tau , n = 3 for pSer262 - Tau , unpaired student ' s t - test , * * p = 0 . 0027 , * p = 0 . 0120 ) ( C ) Images of PLA signal ( green ) for Hrs / Tau interaction in N2a cells co - transfected with FLAG - Hrs and either mCh or mCh - Rab35 ( red ) , and probed with antibodies against FLAG and total ( DA9 ) or phospho - Tau species ( pSer396 / 404 - Tau , pSer262 - Tau , or pSer202 - Tau ) ; scale bar : 5 μm . ( D ) Rab35 overexpression significantly increases PLA puncta for total , pSer396 / 404 - Tau , pSer262 - Tau but not pSer202 - Tau ( n = 121 - 132 cells for total Tau , 178 - 195 cells for pSer396 / 404 - Tau , 71 - 98 cells for pSer262 - Tau , 25 - 29 cells for pSer202 - Tau ; Mann - Whitney test , * * * * p < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120)  (C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm. (D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001)


Figure 5 ( A , B ) Representative immunoblots ( A ) and quantification of Hrs , TSG101 , CHMP2b , and Rab35 protein levels ( B ) from 14 DIV neurons treated with either DMSO ( CON ) or glucocorticoids ( GC ) . GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs , TSG101 , or CHMP2b ( n = 9 - 10 / condition , unpaired student ' s t - test , * p = 0 . 0221 ) ( C ) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells ( for neurons , n = 9 / condition , unpaired student ' s t - test , * * p = 0 . 0036 ; for N2a cells , n = 6 / condition , unpaired student ' s t - test , * p = 0 . 0138 ) ( E , F ) Representative immunoblots ( E ) and quantification of levels of different Rab proteins ( F ) in hippocampus of GC - treated and CON animals . Protein levels of Rab35 , but not other Rabs , are decreased in GC - treated animals compared to CON ones ( n = 5 animals / condition , unpaired student ' s t - test , * p = 0 . 0257 ) ( G , H ) Immunofluorescence staining of Rab35 ( green ) and DAPI ( blue ) ( G ) showing that Rab35 fluorescence intensity is reduced in hippocampal area ( CA1 ) of GC - treated aniamls ( H ) ( n = 15 slices / condition ; unpaired student ' s t - test , * * * p = 0 . 0003 ) . Data information : All numeric data represent mean ± SEM

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221)  (C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138)  (E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)  (G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM


Figure 6 ( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) in 14 DIV neurons expressing mCh - Rab35 or mCh , treated for 24 h with cycloheximide ( CHX ) or DMSO ( CON ) under GC conditions . GC significantly decreases Tau degradation , whereas Rab35 overexpression blocks this effect ( n = 12 - 14 / condition ; 1 - way ANOVA , Dunnet post - hoc analysis , * pmCh vs mCh GC = 0 , 0319 , * pmCh vs Rab35 GC = 0 , 047 ) ( D , E ) Representative immunoblots ( D of Tau levels ( E ) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP , but not Rab35 ( n = 5 - 6 animals / group , 2 replicates , 2 - way ANOVA , GC x Rab35 interaction F1 , 39 = 10 , 51 p = 0 . 002 ; Sidak posthoc analysis * p = 0 . 0168 ) ( F ) Golgi - based 3D neuronal reconstruction of hippocampal pyramidal neurons ( CA1 area ) ( G ) GC treatment reduces the length of apical dendrites in animals expressing EGFP , but not EGFP - Rab35 ( n = 6 - 7 animals / group ; 6 - 8 neurons / animal , 2 - way ANOVA , GC x Rab35 interaction F1 , 155 = 3 . 969 p = 0 . 0481 , overall GC effect F1 , 155 = 8 . 998 p = 0 . 0031 , Sidak posthoc analysis * * p = 0 . 0021 ) ( H ) GC treatment reduces mature spine density in animals expressing EGFP , but not EGFP - Rab35 ( 2 - way ANOVA , GC x Rab35 interaction F1 , 499 = 12 . 33 p = 0 . 0005 , overall GC effect F1 , 499 = 4 . 373 p = 0 . 0370 , Sidak posthoc analysis * * * p = 0 . 0003 ; n = 6 - 7 animals / group ; 6 - 8 neurons per animal ) ( I , J ) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP - expressing animals ; however , this effect is not seen in EGFP - Rab35 - expressing animals ( 3 - way ANOVA , GC x Rab35 interaction F1 , 4212 = 14 , 926 p < 0 . 0001 , Simple effect analysis , Sidak test for multiple comparisons * p120 = 0 . 012 , * * p140 = 0 . 001 , * * p220 = 0 . 002 , * * * p240 < 0 . 001 , * * * p260 < 0 . 001 , * * p280 = 0 . 001 , * * p300 = 0 . 001 , * p320 = 0 . 021 , * p340 = 0 . 047 , n = 6 - 7 animals / group , 6 - 8 neurons per animal )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 (A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047)  (D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168) (F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area)  (G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021)  (H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal)  (I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal)


Figure 1Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right . Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope - tagged proteins in the anti - Mta1 , Mta2 or Mta3 panels ; and a grey triangle shows the location of IgG bands in the anti - Mbd3 panel . 10 % of the input was loaded into " Input " lanes . Molecular weights in kDa are shown at left . Proteins co - purifying with Mta1 , Mta2 or Mta3 in IP - mass spectrometry experiments in wild type cells ( top ) or Mbd3 - null cells ( bottom ) . Proteins showing significant enrichment with the bait protein are located outside the dotted lines . For all panels the protein being immunoprecipitated is indicated in red , the other MTA proteins in purple , and other NuRD components in blue . Each IP / Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation . Relative enrichment of indicated proteins in MTA pulldowns from wild type ( WT ) or Mbd3 - null ( Mbd3KO ) ES cells , normalised to 2x MTA proteins . NuRD components comprising the remodelling subunit are labelled in blue , those comprising the deacetylase subunit in red . As MTA proteins are the bait in these experiments , they may be isolated more efficiently than their interaction partners . Error bars represent standard deviation from three ( Mta1 and Mta2 ) or six ( Mta3 ) replicate pulldowns .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left.Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation.Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.


Figure 2ChIP - seq peaks identified for Mta1 and Mta2 ( left ) , Mta1 and Mta3 ( middle ) or Mta2 and Mta3 ( right ) are plotted by enrichment for each protein . Peaks called for both proteins are indicated in the overlap . ChIP - seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta - bound sites . Mta1 / 2 / 3 refers to peaks identified in all three ChIP - seq datasets , whereas " Mta1 Only " , " Mta2 Only " or " Mta3 Only " refer to peaks only called for that protein . Average enrichment of density plots are plotted for different subsets of Mta ChIP - seq peaks .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O

Figure 2ChIP-seq peaks identified for Mta1 and Mta2 (left), Mta1 and Mta3 (middle) or Mta2 and Mta3 (right) are plotted by enrichment for each protein. Peaks called for both proteins are indicated in the overlap.ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three ChIP-seq datasets, whereas "Mta1 Only", "Mta2 Only" or "Mta3 Only" refer to peaks only called for that protein.Average enrichment of density plots are plotted for different subsets of Mta ChIP-seq peaks.


Figure 3Western blots of wild type ( Control ) , double or triple - null ES cell nuclear extract was probed with antibodies indicated at right . LaminB1 acts as a loading control . Approximate sizes are indicated in kDa . Phase - contrast images of wild type or Mta123 ∆ ES cells in 2i + LIF conditions . Scale bars represent 100µWestern blots of anti - Chd4 immunoprecipitation of nuclear extract from indicated cell lines ( top ) probed with antibodies indicated at right . For the Gatad2b and Mbd3 blots , arrowheads indicate target proteins , while an asterisk marks a non - specific IgG bands . 10 % of the input was loaded into " Input " lanes . Approximate sizes are indicated in kDa . Western blot of a time course of Mta3 deletion in Mta1 ∆ Mta2 ∆ Mta3Flox / Flox : Cre - ER ( Mta12 ∆ 3F / F ) or Control ( Mta12 ∆ 3F / F without Cre - ER ) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control ( αRNAPII ) . The time course is indicated at the top as Days + tamoxifen . Western blot showing rescue of Mta123 ∆ ES cells by ectopic expression of Mta1 , Mta2 or Mta3 from a transgene ( TG ) . Total RNA Polymerase II acts as a loading control ( αRNAPII ) . For all western blots molecular weight is indicated at left in kDa .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in kDa.Phase-contrast images of wild type or Mta123∆ ES cells in 2i+LIF conditions. Scale bars represent 100µWestern blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. For the Gatad2b and Mbd3 blots, arrowheads indicate target proteins, while an asterisk marks a non-specific IgG bands. 10% of the input was loaded into "Input" lanes. Approximate sizes are indicated in kDa.Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (αRNAPII). For all western blots molecular weight is indicated at left in kDa.


Figure 4Comparison of gene expression in Mta123 ∆ ES cells and wild type ( WT ) ES cells . Each circle represents a gene : red indicates spike - in controls , blue indicates genes that are not differentially expressed to a significant degree , and green indicates differentially expressed genes ( 2404 increased , 1293 decreased ) defined with an adjusted p - value < 0 . 05 and a log2 fold - change > 1 . N = 3 for each genotype . Fold change in gene expression is plotted for different subsets of genes . All genes are plotted in black , while subsets of genes located nearest to ChIP - seq peaks for the indicated proteins + Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated . The number of genes in each of the Mta categories are : all genes ( 32271 ) , all differentially expressed ( n = 3701 ) , Mta1 + 2 + 3 ( n = 1738 ) , Mta1 + 2 ( n = 1460 ) , Mta1 ( n = 1020 ) and Mta2 ( n = 924 ) . Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self - renewing conditions ( 2i ) or after 48 hours in the absence of two inhibitors and LIF ( Diff48 ) . Each point represents a biological replicate . Genes associated with indicated GO terms plotted by fold change in expression in Mta123 ∆ ES cells ( x - axis ) or Mbd3 ∆ ES cells ( y - axis ) . Genes are coloured if they are differentially expressed ( log2 fold - change > 1 and adjusted p - value < 0 . 05 ) in either comparison as indicated . The dotted lines show the fold - change cut - off of 2 . GO - terms were identified using David v . 6 . 8 using a Benjamini score with a cutoff of 0 . 05 . Genes associated with core NuRD peaks , with an MTA - only peak , with a Chd4 peak and an MTA - peak but not an Mbd3 peak ( Chd4 + Mta - Mbd3 genes ) , or with an MTA - peak but not a Chd4 peak ( MTA - only genes ) were divided into quartiles based upon expression levels in wild type cells . Change in gene expression is plotted on the x - axis and calculated significance of the expression change on the y - axis . Those genes significantly misexpressed ( | log2FC | > 1 ; p ≤ 0 . 05 ) are indicated in red , genes showing no significant change are plotted in black . The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype.Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924).Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate.Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 using a Benjamini score with a cutoff of 0.05.Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.


Figure 5Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions ( N2B27 ) . White arrowheads in the Mbd3 ∆ panel indicate pockets of ES - like cells . Scale bars represent 100 µm . Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT - qPCR in Control ( green ) Mta123 ∆ ( magenta ) or Mbd3 ∆ ( blue ) ES cells . N ≥ 3 biological replicates , error bars indicate SEM . Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions ( N2B27 + 10 % serum ) . Scale bars represent 100µm . Expression of indicated genes in embryoid body cultures over time was measured by RT - qPCRComparison of expression data for wild type , Mbd3 ∆ or Mta123 ∆ ES cells in self - renewing ( 2i ) conditions or after 48 hours in differentiation conditions ( 48h N2B27 ) with mouse embryonic single cell RNA - seq data Larger shading encloses biological replicates , smaller circles represent individual cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm.Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM.Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm.Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCRComparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data Larger shading encloses biological replicates, smaller circles represent individual cells.


Figure 6Composite images of representative chimaeric embryos made with control ( Mta1Flox / ∆ Mta2 + / + Mta3Flox / Flox ) or Mta123 ∆ ES cells . ES - derived cells express the Kusabira Orange fluorescent marker . Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells . Arrows indicate examples of K - Orange expressing cells in the mutant embryos . Scale bars = 20µm . ( Top ) Number of K - Orange expressing cells observed in chimaeric embryos obtained using control ES cells , Mta123 ∆ ES cells , or Mta123 ∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene ( Mta123 ∆ + Mta2TG ) . P - values calculated using a two - tailed t - test . ( Middle ) Mean number of K - Orange cells per embryo separated by Sox2 expression . P - values calculated using a two - tailed t - test . ( Bottom ) Number of K - Orange and Caspase - 3 positive cells per embryo . P - values calculated using a one - tailed t - test : * P < 0 . 05 , * * * * P < 0 . 0001 , " ns " = not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm.(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.


Figure 1FP titrations show binding of WT α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and K1647E ( red ) , of α - actinin - 1 _ EF34 mutant E847K / E851K to IQ peptides WT ( green ) and K1647E ( purple ) , of full - length α - actinin - 1 to IQ WT ( o ) , and lack of IQ binding to α - actinin - 1 _ CH1 - CH2 ( + ) and α - actinin - 1 _ EF12 ( x ; see Table 2 for binding parameters and standard errors ) . FP titrations showing binding of wild type α - actinin - 1 _ EF34 to IQ peptides WT ( black ) and I1654A ( purple ) and of ACTN1 _ EF34 mutant F833 to IQ WT ( red ; see Table 2 for binding parameters and standard errors ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1FP titrations show binding of WT α-actinin-1_EF34 to IQ peptides WT (black) and K1647E (red), of α-actinin-1_EF34 mutant E847K/E851K to IQ peptides WT (green) and K1647E (purple), of full-length α-actinin-1 to IQ WT (o), and lack of IQ binding to α-actinin-1_CH1-CH2 (+) and α-actinin-1_EF12 (x; see Table 2 for binding parameters and standard errors). FP titrations showing binding of wild type α-actinin-1_EF34 to IQ peptides WT (black) and I1654A (purple) and of ACTN1_EF34 mutant F833 to IQ WT (red; see Table 2 for binding parameters and standard errors).


Figure 2FP titrations showing binding of apoCaM to IQ peptides WT ( black ) and K1662E ( red ) and of apoCaM mutant E88K to IQ WT ( green ) and IQ peptide K1662E ( purple , " charge inversion " ; see Table 4 for binding parameters and standard errors ) . FP titrations showing binding of apo - CaM to IQ peptides WT ( black ) , I1654A ( red ) , and F1658A ( green ) and of apoCaM mutant F90A to IQ WT ( blue ; see Table 4 for binding parameters and standard errors ) .

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Figure 2FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors). FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).


Figure 3HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A and cultured for 22 - 24h before cell attached patch recording in 110 mM Ba2 + . Single CaV1 . 2 channel recordings of CaV1 . 2 WT and K1647A . Holding potential ( HP ) was - 80 mV and test potential ( TP ) 0 mV . Shown are 10 consecutive sweeps from representative experiments ( see Fig . EV 4 for more sweeps ) . Dot plots and means + SEM for availability ( i . e . , likelihood that a sweep had at least one event ; c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( e ) , and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 , * * * p < 0 . 001 compared to WT ; one - way ANOVA with Bonferroni post - hoc test ( c ) or Welch ANOVA with Tamhane T2 test ( d - g ) ; n = 7 to 35 see Table 5 for more details ) .

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Figure 3HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+. Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was -80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps). Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).


Figure 4HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A plus , if indicated , α - actinin - 1 before cell attached patch recording . Representative single CaV1 . 2 channel recordings of WT CaV1 . 2 alone or with WT or E847K / E851K mutant α - actinin - 1 . Mean assemble averages for all experiments for each combination . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and the mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 001 ; unpaired t - test ; n = 13 to 35 ; see Table 6 for more details ) .

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Figure 4HEK293 cells were transfected with α11.2, α2δ-1, and β2A plus, if indicated, α-actinin-1 before cell attached patch recording. Representative single CaV1.2 channel recordings of WT CaV1.2 alone or with WT or E847K/E851K mutant α-actinin-1. Mean assemble averages for all experiments for each combination. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and the mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (*p<0.05, **p<0.01, ****p<0.001; unpaired t-test; n = 13 to 35; see Table 6 for more details).


Figure 5HEK293 cells were transfected with α11 . 2 with the K1647E mutation , α2δ - 1 , and β2A plus α - actinin - 1 WT or E847K / E851K before cell attached patch recording . Representative single CaV1 . 2 channel recordings of CaV1 . 2 K1647E with WT or E847K / E851K α - actinin - 1 . Mean assemble averages for all experiments for both combinations . Dot plots and means + SEM for availability ( c ) , NPo ( d ) , Po ( e ) , unitary current amplitude i ( f ) , and mean of the current of a single channel calculated from the ensemble averages of each experiment ( g ) ( * * p < 0 . 01 ; unpaired t - test ; n = 16 to 30 ; see Suppl . Fig . 8 and Table 6 for more details ) .

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Figure 5HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording. Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1. Mean assemble averages for all experiments for both combinations. Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).


Figure 6HEK293 cells were transfected with WT α11 . 2 , α2δ - 1 , and β2A + WT α - actinin - 1 and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of NeutrAvidin pull - down samples ( from lysate containing 600 μg protein ) and total lysate samples containing 20 μg protein . α11 . 2 was detected with an antibody against its HA tag ( top ) , which is present in all constructs used throughout this work . Pull - down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull - down samples . Immunoblotting for vinculin ( middle ) and tubulin ( bottom ) indicated that comparable amounts of these intracellular control proteins were present in lysate samples . Their absence in pull - down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling of control α11 . 2 samples lacking α - actinin - 1 co - expression ( mean set to 100 % ; see Methods ; * * p < 0 . 01 two - tailed t - test ; n = 7 ) . HEK293 cells were transfected with WT or K1647E mutant α11 . 2 , α2δ - 1 , and β2A , and WT or E847K / E851K mutant α - actinin - 1 , or CFP alone as negative control and cultured for 22 - 24h before surface biotinylation . Shown are representative immunoblots of pull - down and lysate samples as in ( a ) . Bar graph shows means + SEM of the pull - down immunosignals in mutants relative to surface labelling in the WT / WT control ( mean set to 100 % ; see Methods ) . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with Tukey post hoc test ; n = 4 - 5 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6HEK293 cells were transfected with WT α11.2, α2δ-1, and β2A + WT α-actinin-1 and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull-down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α11.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull-down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull-down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull-down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling of control α11.2 samples lacking α-actinin-1 co-expression (mean set to 100%; see Methods; **p<0.01 two-tailed t-test; n = 7).HEK293 cells were transfected with WT or K1647E mutant α11.2, α2δ-1, and β2A, and WT or E847K/E851K mutant α-actinin-1, or CFP alone as negative control and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of pull-down and lysate samples as in (a). Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling in the WT/WT control (mean set to 100%; see Methods). **p<0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc test; n = 4-5).


Figure 7HEK293 cells were transfected with α11 . 2 , α2δ - 1 , and β2A before whole cell patch recording in 20 mM Ba2 + . Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of - 80 mV to the indicated potentials ( the voltage protocol is schematized in the upper left corner ) . Representative current traces upon step depolarizations to the reversal potential ( Erev ) for 20 ms to obtain movement of the ON - gating charges ( Qon ) , and subsequent to - 50 mV for 10 ms to obtain tail currents ( Itail ) . Insets : magnifications of exemplary Qon for CaV1 . 2 WT , K1647A , Y1649A , I1654A , F1658A and K1662E . Plots of Itail ( in this panel corrected for variations in cell capacitance ) versus total detectable charge transfer for Qon . Slopes of regression curves are strongly reduced for CaV1 . 2 K1647A , Y1649A and I1654A versus WT ( see Table 7 for more details ) . Dot plots and means + SEM of Qon ( in this panel corrected for variations in cell capacitance ; * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with Bonferroni post - hoc test ; n = 13 - 18 ; see Table 7 for more details ) . The reduction in slope of the regression curve of combined population data for K1647A , Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α - actinin binding to the IQ motif is diminished .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+. Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner). Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E. Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details). Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details). The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.


Figure 1 ( C ) Fluorescence polarization assays demonstrating that a peptide containing the BH3 ‐ like domain of Beclin ‐ 1 interacts with Bcl ‐ XL . Recombinant Bcl ‐ XL ΔTM was incubated with carboxyfluorescein ‐ labeled Bak BH3 peptide , in the absence or presence of wild ‐ type ( WT ) Beclin ‐ 1 BH3 or either of the two mutant peptides described in B . ( D ) Affinity determination of the interaction between recombinant Bcl ‐ XLΔTM and fluorescent Beclin ‐ 1 BH3 , by fluorescence polarization . A comparison between WT Bcl ‐ XL ( squares ) and G138A Bcl ‐ XL ( triangles ) is shown ( means ± s . d . , n = 3 separate experiments ) . ( E ) Co ‐ immunoprecipitation of Bcl ‐ XL and WT or mutant Beclin ‐ 1 . HeLa cells were transfected with the indicated constructs , followed by immunoprecipitation of Flag ‐ tagged Bcl ‐ XL and immunochemical detection of Beclin ‐ 1 . ( F ) Co ‐ immunoprecipitation of Beclin ‐ 1 with ‐ WT and mutant ( G138A ) Bcl ‐ XL . Results are representative of at least three experiments yielding similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B.(D) Affinity determination of the interaction between recombinant Bcl‐XLΔTM and fluorescent Beclin‐1 BH3, by fluorescence polarization. A comparison between WT Bcl‐XL(squares) and G138A Bcl‐XL (triangles) is shown (means±s.d., n=3 separate experiments).(E) Co‐immunoprecipitation of Bcl‐XL and WT or mutant Beclin‐1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag‐tagged Bcl‐XL and immunochemical detection of Beclin‐1.(F) Co‐immunoprecipitation of Beclin‐1 with‐WT and mutant (G138A) Bcl‐XL. Results are representative of at least three experiments yielding similar results.


Figure 2 ( A ) Competition between Beclin ‐ 1 BH3 and ABT737 for Bcl ‐ XL binding . A fluorescent 25 ‐ mer peptide containing the BH3 ‐ like domain of Beclin ‐ 1 ( Figure 1D ) was docked to recombinant Bcl ‐ XL ΔTM protein in the absence or presence of ABT737 . The IC50 of ABT737 , as measured in the presence of 15 nM of peptide and 100 nM of Bcl ‐ XL ΔTM , was 1 . 7 μM . ( B , C ) Abolition of the interaction between Bcl ‐ XL and Beclin ‐ 1 by ABT737 in intact cells . Co ‐ immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E . Sixteen hours before the immunoprecipitation , cells were exposed to ABT737 . Similar results were obtained for co ‐ transfected Bcl ‐ XL and Beclin ‐ 1 ( B ) and for endogenous Beclin ‐ 1 interacting with Flag ‐ tagged Bcl ‐ XL ( C ) . ( D , E ) Differential effect of ABT737 on the interaction between Bcl ‐ 2 ( D ) or Mcl ‐ 1 ( E ) and Beclin ‐ 1 . This experiment was designed as ( B ) . All experiments have been performed at least three times , with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Competition between Beclin‐1 BH3 and ABT737 for Bcl‐XL binding. A fluorescent 25‐mer peptide containing the BH3‐like domain of Beclin‐1 ( Figure 1D) was docked to recombinant Bcl‐XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl‐XL ΔTM, was 1.7 μM.(B, C) Abolition of the interaction between Bcl‐XL and Beclin‐1 by ABT737 in intact cells. Co‐immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co‐transfected Bcl‐XL and Beclin‐1 (B) and for endogenous Beclin‐1 interacting with Flag‐tagged Bcl‐XL (C).(D, E) Differential effect of ABT737 on the interaction between Bcl‐2 (D) or Mcl‐1 (E) and Beclin‐1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.


Figure 3 ( A , B ) Detection of autophagic vacuoles by LC3 ‐ GFP and their modulation by ABT737 and by Beclin ‐ 1 ‐ specific siRNAs . HeLa cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , 24 h later re ‐ transfected with LC3 ‐ GFP , cultured in complete medium ( CM ) for 24 h , and finally kept 12 h either in CM or in nutrient ‐ free ( NF ) conditions , in the presence or absence of 1 μM ABT737 . Representative microphotographs of cells cultured in NF medium are shown in ( A ) and the percentage ( means ± s . d . , n = 3 separate experiments ) of LC3 ‐ GFP ‐ transfected cells bearing LC3 ‐ GFP aggregates in the cytoplasm ( LC3 ‐ GFPvac ) are quantified in ( B ) . The insert in ( B ) demonstrates the efficiency of the Beclin ‐ 1 ‐ specific siRNAs , as quantified by immunoblot . ( C , D ) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate ( CMFDA ) . Cells were transfected with control or Beclin ‐ 1 ‐ specific siRNAs , cultured for 48 h in CM , washed , cultured in CM ( D ) or NF ( C , D ) for 12 h , stained with CMFDA , and either photographed ( C ) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole ( arrow head ) ( means ± s . d . , n = 3 separate experiments ) . ( E ) Ultrastructure of autophagic vacuoles induced by ABT737 . Transmission electron microphotographs are shown . ( F ) Detection of dead and dying cells in the cultures . Cells treated as in ( A ) were stained with the ΔΨm ‐ sensitive dye DiOC6 ( 3 ) and the vital dye propidium iodide ( PI ) . The black portions of the columns refer to the DiOC6 ( 3 ) low PI + population ( dead ) and the remaining part of the column corresponds to the DiOC6 ( 3 ) low PI − ( dying ) population . Results are means ± s . d . of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot.(C, D) Detection of cytoplasmicvacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin‐1‐specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmicvacuole (arrow head) (means±s.d., n=3 separate experiments).(E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown.(F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm‐sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments.


Figure 4Quantification of autophagic vacuoles induced by ABT737 . HeLa cells transfected with the indicated siRNAs ( specific for Emerin or for Beclin ‐ 1 at 0 h ) were re ‐ transfected with LC3 ‐ GFP finally cultured in nutrient ‐ free ( NF ) conditions ( 60 - 72 h ) , in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature ( AV1 ) or mature ( AV2 ) autophagic vacuoles . Representative pictures are shown in ( A ) . The number of AV1 and AV2 was determined for a minimum of 50 cells ( means ± s . e . m . ) ( B ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60-72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopy detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A).The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).


Figure 5 ( A - D ) Modulation of Beclin ‐ 1 ‐ induced autophagy by BH3 mutants and ABT737 . Cells were transfected with GFP ‐ LC3 together with an empty control vector or Beclin ‐ 1 ( WT , L116A , F123A ) and cultured for 48 h , followed by overnight culture in CM ( B ) or NF ( A , B ) in the absence or presence of 1 μM ABT737 . Representative microphotographs are shown in ( A ) and the LC3 ‐ GFP ‐ positive vacuoles per cell are quantified in ( B ) ( means ± s . d . ; n = 3 separate experiments ) . Alternatively , cells were not transfected and stained with CMFDA to detect vacuoles , as shown in ( C ) and ( D ) , yielding similar results as for the LC3 ‐ GFP method . ( E ) BH3 ‐ dependent modulation of Beclin ‐ 1 ‐ induced autophagy by anti ‐ apoptotic Bcl ‐ 2 proteins . Cells were transfected simultaneously with Beclin ‐ 1 ( or control vector ) and equivalent amounts of plasmids coding for WT Bcl ‐ XL , Bcl ‐ XL G138A , Bcl ‐ 2 or Mcl ‐ 1 ( or control vector ) , as well as GFP ‐ LC3 . The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h ‐ long experiment . The percentage of cells exhibiting the accumulation of LC3 ‐ GFP in vacuoles ( LC3 ‐ GFPvac ) is quantified as means ± s . d . ( n = 3 separate experiments ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method.(E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).


Figure 6 ( A ) Immunoprecipitation of endogenous Bcl ‐ 2 and Beclin ‐ 1 . Untransfected HeLa or MV4 . 11 cells were treated by culture in nutrient ‐ free EBSS or with ABT737 ( 1 μM ) . ( B ) Immunoprecipitation of wild ‐ type and ER ‐ targeted Bcl ‐ 2 with Beclin ‐ 1 . Cells stably transfected with wild type , ER ‐ and mitochondrion ‐ targeted Bcl ‐ 2 were subjected to starvation or treated with ABT737 , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 . ( C , D ) Immunoprecipitation of Bcl ‐ 2 and Beclin ‐ 1 in subcellular fractions . Cells treated as in ( B ) were lysed and subjected to subcellular fractionation to enrich either ER vesicles ( microsomes , ( C ) ) or mitochondria ( heavy membrane fraction , ( D ) ) , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Beclin ‐ 1 . Calreticulin and Hsp60 were used as internal controls . Results are representative of three independent determinations .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunoprecipitation of endogenous Bcl‐2 and Beclin‐1. Untransfected HeLa or MV4.11 cells were treated by culture in nutrient‐free EBSS or with ABT737 (1 μM).(B) Immunoprecipitation of wild‐type and ER‐targeted Bcl‐2 with Beclin‐1. Cells stably transfected with wild type, ER‐ and mitochondrion‐targeted Bcl‐2 were subjected to starvation or treated with ABT737, followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1.(C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.


Figure 7 ( A , B ) Interactions between Bcl ‐ XL , Beclin ‐ 1 and Bad in conditions of autophagy induction . Cells were either treated by nutrient depletion ( A ) or addition of 1 μM rapamycin , 1 mM lithium chloride , 100 μM L ‐ 690 , 330 or 50 μM carbamazepine ( B ) , followed by immunprecipitation of Bcl ‐ XL ( as in Figure 2C ) and revealing the immunoblots by antibodies specific for Bcl ‐ XL , Beclin ‐ 1 or Bad . ( C ) Interaction between endogenous Bad and Bcl ‐ 2 . HeLa cells were treated with ABT737 ( 1 μM ) or nutrient ‐ depleted , followed by immunoprecipitation of Bcl ‐ 2 and immunodetection of Bad . ( D , E ) Impact of Bad depletion on autophagy . Cells were transfected with LC3 ‐ GFP together with siRNAs specific for Bad , siRNAs specific for emerin or the Beclin ‐ 1 ‐ associated PI3 kinase Vps34 . Forty ‐ eight hours later , when the siRNAs had down ‐ regulated the proteins of interest ( D ) , cells were subjected by nutrient depletion ( NF ) or addition of rapamycin ( E ) and the frequency of cells exhibiting LC3 ‐ GFP aggregation as a marker of autophagy was assessed after 18 h ( means ± s . d . , * P0 . 05 , n = 3 separate experiments ) . ( F ) Effect of the Bad knock ‐ out on autophagy . Wild Type ( WT ) or Bad − / − MEF were transfected with LC3 ‐ GFP and then subjected to nutrient depletion ( NF ) and / or treatment with ABT737 ( 18h ; means ± s . d . , n = 3 ) . The asterisks denotes a significant ( P0 . 05 ) effect of Bad deficiency . ( G ) Effect of the Bad knockout on the Beclin ‐ 1 / Bcl ‐ 2 interaction . WT or Bad − / − MEF were subjected to the indicated treatments , followed by immunoprecipitation of Bcl ‐ 2 and immunobloting of Beclin ‐ 1 . ( H ) Overexpression of Bad triggers autophagy . Cells were transfected with LC3 ‐ GFP alone ( Control ) , together with vector ‐ only or with a vector encoding Bad , and subjected 48 h later to nutrient depletion ( NF ) . Cells were cultured in the continuous presence or absence of the pan ‐ caspase inhibitor Z ‐ VAD ‐ fmk ( 50 μM ) for 16 h , followed by assessment of autophagy as in ( E ) ( means ± s . d . , n = 3 separate experiments , * P0 . 01 ) . Immunoblots are representative of at least three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad.(C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were treated with ABT737 (1 μM) or nutrient‐depleted, followed by immunoprecipitation of Bcl‐2 and immunodetection of Bad.(D, E) Impact of Bad depletion on autophagy. Cells were transfected with LC3‐GFP together with siRNAs specific for Bad, siRNAs specific for emerin or the Beclin‐1‐associated PI3 kinase Vps34. Forty‐eight hours later, when the siRNAs had down‐regulated the proteins of interest (D), cells were subjected by nutrient depletion (NF) or addition of rapamycin (E) and the frequency of cells exhibiting LC3‐GFP aggregation as a marker of autophagy was assessed after 18 h (means±s.d., *P0.05, n=3 separate experiments).(F) Effect of the Bad knock‐out on autophagy. Wild Type (WT) or Bad−/− MEF were transfected with LC3‐GFP and then subjected to nutrient depletion (NF) and/or treatment with ABT737 (18h; means±s.d., n=3). The asterisks denotes a significant (P0.05) effect of Bad deficiency.(G) Effect of the Bad knockout on the Beclin‐1/Bcl‐2 interaction. WT or Bad−/− MEF were subjected to the indicated treatments, followed by immunoprecipitation of Bcl‐2 and immunobloting of Beclin‐1.(H) Overexpression of Bad triggers autophagy. Cells were transfected with LC3‐GFP alone (Control), together with vector‐only or with a vector encoding Bad, and subjected 48 h later to nutrient depletion (NF). Cells were cultured in the continuous presence or absence of the pan‐caspase inhibitor Z‐VAD‐fmk (50 μM) for 16 h, followed by assessment of autophagy as in (E) (means±s.d., n=3 separate experiments, *P0.01). Immunoblots are representative of at least three independent experiments.


Figure 8 ( A ) Representative images of wt and egl ‐ 1 mutant embryos , bearing the plgg − 1DsRED ( B ) Quantification of LGG ‐ 1 ∷ DsRED fluorescence as determined in ( A ) . Each point represents the measurement for one individual embryo . Horizontal bars denote means and gray areas denote standard errors .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Representative images of wt and egl‐1 mutant embryos, bearing the plgg−1DsRED(B) Quantification of LGG‐1∷DsRED fluorescence as determined in (A). Each point represents the measurement for one individual embryo. Horizontal bars denote means and gray areas denote standard errors.


Figure 1Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . Volcano plots showing the log fold - change ( X - axis ) and uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are given for ( A ) 48 antihypertensive drugs The enrichment of positive / negative ACE2 expression regulators in different drug classes based on their mechanism of action ( MOA ) was tested with the GSEA method as implemented in the R package fgsea ( Segushichev , 2016 ; Methods ) , and the enrichment significance ( negative log10 P values ) is shown in bar plots in ( B ) , for the analysis on the 48 antihypertensive drugs CCBs , calcium channel blockers ; ARBs , angiotensin II type - I receptor blockers ; ACEIs , angiotensin - converting enzyme inhibitorsDifferential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . ( C ) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP ( Subramanian et al , 2017 ) . Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . The ACE2 expression levels in control and drug - treated groups for the top significant drugs ( vorinostat , panobinostat , and isotretinoin ) among the 672 clinically approved drugs are shown with boxplots in ( D ) , the ACE2 expression log fold - change values and adjusted P values from limma , as well as the number of samples per group are labeled for each drug . In the boxplot of panel ( D ) , the center line , box edges and whiskers denotes the median , interquartile range and the rest of the distribution in respective order , except for points that were determined to be outliers using a method that is a function of the interquartile range , as in standard box plots . Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . The enrichment of positive / negative ACE2 expression regulators in different drug classes based on their mechanism of action ( MOA ) was tested with the GSEA method as implemented in the R package fgsea ( Segushichev , 2016 ; Methods ) , and the enrichment significance ( negative log10 P values ) is shown in bar plots in ( E ) , for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines ( 16 cancer and 12 normal cells ; this list is provided in Table EV2A with details including primary site , subtype and donor demographics ) where each drug may have been tested on a different subset of cells ( Methods ) . and performed enrichment analysis using the WHO ATC drug classification data ( World Health Organization , 2006 ) , and top enriched drug classes are shown in ( F ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are given for (A) 48 antihypertensive drugs The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (B) , for the analysis on the 48 antihypertensive drugs CCBs, calcium channel blockers; ARBs, angiotensin II type-I receptor blockers; ACEIs, angiotensin-converting enzyme inhibitorsDifferential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). (C) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP (Subramanian et al, 2017).Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The ACE2 expression levels in control and drug-treated groups for the top significant drugs (vorinostat, panobinostat, and isotretinoin) among the 672 clinically approved drugs are shown with boxplots in (D), the ACE2 expression log fold-change values and adjusted P values from limma, as well as the number of samples per group are labeled for each drug. In the boxplot of panel (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots.Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (E), for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines (16 cancer and 12 normal cells; this list is provided in Table EV2A with details including primary site, subtype and donor demographics) where each drug may have been tested on a different subset of cells (Methods). and performed enrichment analysis using the WHO ATC drug classification data (World Health Organization, 2006), and top enriched drug classes are shown in (F).


Figure 2 ( A - D ) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database , spanning both cancer and non - cancer datasets ( Methods ) . Volcano plots showing the log fold - change ( X - axis ) and uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are displayed in ( A ) for 74 datasets of cells or tissue samples from lung , involving 42 clinically approved drugs , and in ( C ) for 35 kidney datasets involving 29 clinically approved drugs . Among these , we focused on the top significant drugs that modulate ACE2 expression in non - cancerous cells or tissue samples from lung and kidney , which are shown in ( B ) and ( D ) , respectively , where the ACE2 expression ( Y - axis ) difference between control and treated groups ( X - axis ) are shown with boxplots . In the title of each boxplot , the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided . Different datasets involving experiments using the same drug were analyzed and presented separately , since the sample types can be different across datasets . All the P values are computed from differential expression analysis using limma ( Ritchie et al , 2015 ) ( Methods ) . In the boxplots of panels ( B ) and ( D ) , the center line , box edges and whiskers denotes the median , interquartile range and the rest of the distribution in respective order , except for points that were determined to be outliers using a method that is a function of the interquartile range , as in standard box plots . Abbreviations : HBEC , human primary bronchial epithelial cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-D) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database, spanning both cancer and non-cancer datasets (Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are displayed in (A) for 74 datasets of cells or tissue samples from lung, involving 42 clinically approved drugs, and in (C) for 35 kidney datasets involving 29 clinically approved drugs. Among these, we focused on the top significant drugs that modulate ACE2 expression in non-cancerous cells or tissue samples from lung and kidney, which are shown in (B) and (D), respectively, where the ACE2 expression (Y-axis) difference between control and treated groups (X-axis) are shown with boxplots. In the title of each boxplot, the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided. Different datasets involving experiments using the same drug were analyzed and presented separately, since the sample types can be different across datasets. All the P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). In the boxplots of panels (B) and (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. Abbreviations: HBEC, human primary bronchial epithelial cells.


Figure 1 ( A ) Recombinant c10orf76 directly binds to PI4KB in vitro . His - pulldown assays of baculovirus / Sf9 produced 6xHis - c10orf76 ( 3 μM ) were carried out with untagged PI4KB ( 2 . 5 μM ) . ( B ) PI4KB and c10orf76 form a stable complex . The complex of c10orf76 - PI4KB eluted from a S200 superdex 10 / 300 GL increase gel filtration column ( GE ) at a volume consistent with a heterodimer ( 169 kDa ) , while c10orf76 alone eluted at a volume consistent with a monomer ( 79 kDa ) . Lines with MW values indicate elution of MW standards ( 158 kDa aldolase , 75 kDa conalbumin ) . ( C ) PI4KB is potently inhibited by c10orf76 in a dose - dependent manner in vitro . Kinase assays of PI4KB ( 20 nM ) in the presence of varying concentrations of c10orf76 ( 1 . 6 nM - 1 μM ) were carried out on pure PI lipid vesicles ( 0 . 5 mg / L ) in the presence of 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . IC50 values were determined by one binding site , nonlinear regression ( curve fit ) using Graphpad . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( D ) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition . Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles ( 0 . 5 mg / mL ) with 100 μM ATP , and Golgi mimic vesicles ( 0 . 5 mg / ml , 10 % PS , 20 % PI , 25 % PE , 45 % PC ) with 10 μM ATP . PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively , with c10orf76 present at 500 nM in both experiments . The data is normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( E ) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N - lobe linker and smaller changes in the helical domain and C - lobe of the kinase domain . The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB ( 400 nM ) in the presence and absence of c10orf76 ( 400 nM ) over all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) . Each dot represents a peptide graphed on the x - axis according to the central residue . The red boxes highlight key regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( G ) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB . H / D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 ( 400 nM ) in the presence of PI4KB ( 400 nM ) at all time points ( 3s at 1 oC ; 3s , 30s , and 300s at 23 oC ) analyzed . Red boxes highlight regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , and unpaired two - tailed student t - test p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( H ) The PI4KB N - lobe linker undergoes a disorder - to - order transition upon binding c10orf76 . Selected peptides ( including the sequence , domain information , and numbering ) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time . Error bars represent standard deviation of independent technical replicates ( n = 3 ) , and are typically smaller than the size of the point on the graph .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM).(B) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).(H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.