Figure 1Senescence - associated β - galactosidase ( SA - β - gal ) staining in A549 , HAFF , HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses ( shCtrl ) or those targeting GUARDIN ( shGUARDIN ) . The graph shows percentage of SA - β - gal - positive cells . Senescence - associated heterochromatin foci formation ( SAHF ) in A549 cells with either shCtrl or shGUARDIN . Representative IF staining with H3K9me3 antibodies ( green ) and nuclear Hoechst counterstaining ( blue ) . Secretion of SASP factors IL - 6 and IL - 8 in A549 cells with either shCtrl or shGUARDIN . Western blotting for p21 , p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN - 1 , - 2 , respectively ( left ) . Knockdown efficiencies analyzed by qPCR ( right ) . SA - β - gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA ( shp21 ) . The graph shows percentage of SA - β - gal - positive cells . qPCR measurement of p21 mRNA in A549 , HepG2 , HAFF and H1299 cells with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) . Western blotting for p53 and p21 in A549 , HepG2 , HAFF and H1299 cells with either GUARDIN shRNA knockdown ( top ) or GUARDIN overexpression ( bottom ) . qPCR assays of NOXA , TIGAR , BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN .

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Figure 1Senescence-associated β-galactosidase (SA-β-gal) staining in A549, HAFF, HepG2 and H1299 cells after 48h transduction with either negative control lentiviruses (shCtrl) or those targeting GUARDIN (shGUARDIN). The graph shows percentage of SA-β-gal-positive cells.Senescence-associated heterochromatin foci formation (SAHF) in A549 cells with either shCtrl or shGUARDIN. Representative IF staining with H3K9me3 antibodies (green) and nuclear Hoechst counterstaining (blue).Secretion of SASP factors IL-6 and IL-8 in A549 cells with either shCtrl or shGUARDIN.Western blotting for p21, p16 and p15 in HepG2 cells after 48h transduction with shCtrl or shGUARDIN-1, -2, respectively (left). Knockdown efficiencies analyzed by qPCR (right).SA-β-gal staining in A549 cells after 24h transduction with either shCtrl or shGUARDIN in combination with p21 shRNA (shp21). The graph shows percentage of SA-β-gal-positive cells.qPCR measurement of p21 mRNA in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).Western blotting for p53 and p21 in A549, HepG2, HAFF and H1299 cells with either GUARDIN shRNA knockdown (top) or GUARDIN overexpression (bottom).qPCR assays of NOXA, TIGAR, BAX and p21 mRNA levels in A549 cells with either shCtrl or shGUARDIN.


Figure 2SDS - PAGE of RNA pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - cell lysates of A549 cells indicating putative GUARDIN - binding proteins ( left ) ; protein identities with high probabilities were determined by mass spectrometry ( right ) . RNA pulldown assays interrogating putative GUARDIN - associated proteins identified in ( A ) from whole - cell lysates of A549 and HAFF cells . BRCA1 , BARD1 served as positive controls and β - actin served as negative controls . RNA immunoprecipitation ( RIP ) assays against IgG / LRP130 antibodies in whole - cell lysates of A549 cells . Subcellular localization of GUARDIN and its co - localization with LRP130 . RNA FISH for GUARDIN ( red ) and IF for LRP130 ( green ) in A549 cells with either shCtrl or shGUARDIN . Nucleus were counterstained with Hoechst ( blue ) . RNA pulldown assays using biotin - labelled sense / antisense probes against GUARDIN from whole - cell lysates of A549 cells . GUARDIN levels were measured by RT - PCR and co - precipitated LRP130 and PGC1α detected by Western blotting . BRCA1 and β - actin served as positive and negative controls , respectively . RIP assay using IgG / PGC1α antibodies from whole - cell lysates of A549 cells . GUARDIN , LRP130 and PGC1α levels were measured as per ( E ) . Two - step IP assays in whole - cell lysates of A549 cells transfected with FLAG - tagged PGC1α . First phase IPs were conducted with FLAG antibodies ( left ) and following elution with FLAG peptides , eluates were further subjected to second phase IPs with LRP130 antibodies ( right ) . Samples were subjected to Western blotting and qPCR analysis for LRP130 , PGC1α and GUARDIN , respectively . Co - immunoprecipitation ( co - IP ) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN . LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130 , PGC1α and β - actin as loading control . Mammalian two - hybrid assays between pACT - LRP130 and pBIND - PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN . Samples were subjected to the luciferase activity assays . LRP130 / PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 ( top left ) or shPGC1α ( bottom left ) as indicated . qPCR assays for p21 mRNA levels were performed in parallel ( right panels ) . Western blotting analysis of LRP130 , PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN .

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Figure 2SDS-PAGE of RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells indicating putative GUARDIN-binding proteins (left); protein identities with high probabilities were determined by mass spectrometry (right).RNA pulldown assays interrogating putative GUARDIN-associated proteins identified in (A) from whole-cell lysates of A549 and HAFF cells. BRCA1, BARD1 served as positive controls and β-actin served as negative controls.RNA immunoprecipitation (RIP) assays against IgG/LRP130 antibodies in whole-cell lysates of A549 cells.Subcellular localization of GUARDIN and its co-localization with LRP130. RNA FISH for GUARDIN (red) and IF for LRP130 (green) in A549 cells with either shCtrl or shGUARDIN. Nucleus were counterstained with Hoechst (blue).RNA pulldown assays using biotin-labelled sense/antisense probes against GUARDIN from whole-cell lysates of A549 cells. GUARDIN levels were measured by RT-PCR and co-precipitated LRP130 and PGC1α detected by Western blotting. BRCA1 and β-actin served as positive and negative controls, respectively.RIP assay using IgG/PGC1α antibodies from whole-cell lysates of A549 cells. GUARDIN, LRP130 and PGC1α levels were measured as per (E).Two-step IP assays in whole-cell lysates of A549 cells transfected with FLAG-tagged PGC1α. First phase IPs were conducted with FLAG antibodies (left) and following elution with FLAG peptides, eluates were further subjected to second phase IPs with LRP130 antibodies (right). Samples were subjected to Western blotting and qPCR analysis for LRP130, PGC1α and GUARDIN, respectively.Co-immunoprecipitation (co-IP) between LRP130 and PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. LRP130 was precipitated and samples subjected to Western blotting analysis for LRP130, PGC1α and β-actin as loading control.Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells after 48h transduction with shCtrl or shGUARDIN. Samples were subjected to the luciferase activity assays.LRP130/PGC1α and p21 protein expression was measured by Western blot in A549 and HAFF cells after 48h transduction with shCtrl or shLRP130 (top left) or shPGC1α (bottom left) as indicated. qPCR assays for p21 mRNA levels were performed in parallel (right panels).Western blotting analysis of LRP130, PGC1α and p21 protein levels in HAFF and A549 cells after 48h transduction with shCtrl or shGUARDIN.


Figure 3ChIP assays detecting binding of LRP130 / PGC1α to the p21 promoter using qPCR and RT - PCR ( top left and right , respectively ) . IgG and p53 served as a negative and positive controls , respectively . Schematic illustrations of the putative LRP130 / PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter ( bottom ) . qPCR assays for GUARDIN , FOXO1 , FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN . Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4 . β - actin served as loading control . Half - life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg / ml of actinomycin ( ActD ) for the indicated times . ChIP assays detecting binding of LRP130 / PGC1α to putative binding sites in the FOXO4 promoter using qPCR ( top ) . Data were normalized to the IgG negative control . Schematic illustration of the LRP130 / PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter ( bottom ) . Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids . qPCR assays for LRP130 and FOXO4 mRNA levels ( upper ) and Western blotting analysis for LRP130 protein level ( lower ) in A549 cells after 48h transduction with LRP130 overexpression or knockdown . qPCR assays for PGC1α and FOXO4 mRNA level ( upper ) and Western blotting analysis for PGC1α protein level ( lower ) in A549 cells after 48h transduction with PGC1α overexpression or knockdown . Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids .

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Figure 3ChIP assays detecting binding of LRP130/PGC1α to the p21 promoter using qPCR and RT-PCR (top left and right, respectively). IgG and p53 served as a negative and positive controls, respectively. Schematic illustrations of the putative LRP130/PGC1α binding sites within DNase I hypersensitive regions of the p21 promoter (bottom).qPCR assays for GUARDIN, FOXO1, FOXO3a and FOXO4 in A549 cells after 24h transduction with shCtrl or shGUARDIN.Western blotting assays for FOXO4 and p21 protein expression in A549 cells after 48h transduction with shCtrl or shGUARDIN alone or in combination with shFOXO4. β-actin served as loading control.Half-life times of FOXO4 mRNA in A549 cells with shCtrl or shGUARDIN measured by qPCR after treating cells with 5 μg/ml of actinomycin (ActD) for the indicated times.ChIP assays detecting binding of LRP130/PGC1α to putative binding sites in the FOXO4 promoter using qPCR (top). Data were normalized to the IgG negative control. Schematic illustration of the LRP130/PGC1α binding sites within DNase I hypersensitive regions of the FOXO4 promoter (bottom).Luciferase assays conducted in A549 cells transfected with LRP130 or PGC1α using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.qPCR assays for LRP130 and FOXO4 mRNA levels (upper) and Western blotting analysis for LRP130 protein level (lower) in A549 cells after 48h transduction with LRP130 overexpression or knockdown.qPCR assays for PGC1α and FOXO4 mRNA level (upper) and Western blotting analysis for PGC1α protein level (lower) in A549 cells after 48h transduction with PGC1α overexpression or knockdown.Luciferase activity assays in A549 cells transduced with shCtrl or shGUARDIN using the pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.


Figure 4qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24hqPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times ( B ) . Western blotting assays for p53 , LRP130 , FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle ( DMSO ) for 48h . A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h . Western blotting was used to measure p21 levels ( left ) while conducting SA - β - gal staining in parallel ( middle ) with the percentage of senescent cells calculated from the SA - β - gal staining ( right ) . Mammalian two - hybrid assays between pACT - LRP130 and pBIND - PGC1α in A549 cells treated with DMSO or 100nM rapamycin . Samples were subjected to the luciferase activity assays . SIX4 and FOSL2 mRNA ( left ) and protein levels ( right ) measured by qPCR and Western blotting , respectively in A549 cells treated with DMSO or 100nM rapamycin . TBP served as a control . qPCR ( upper ) and Western blotting ( lower ) assays for GUARDIN , SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 ( left ) , shFOSL2 ( middle ) or FOSL2 ( right ) after transduction for 48h . Luciferase assays in A549 cells transduced with shCtrl , shSIX4 or shFOSL2 using pGL3 ( negative control ) or pGL3 - FOXO4 promoter reporter plasmids . ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT - PCR assay . UPe and UPk served as a positive and negative controls , respectively [ 54 ] .

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Figure 4qPCR assays for GUARDIN expression in A549 cells treated with increasing doses rapamycin for 24hqPCR assays for GUARDIN expression in A549 cells treated with 100nM of rapamycin for the indicated times (B).Western blotting assays for p53, LRP130, FOXO4 and p21 expression in A459 and H1299 cells treated with 100nM of rapamycin or vehicle (DMSO) for 48h.A549 cells with shCtrl or shGUARDIN were treated with 100nM rapamycin or DMSO vehicle in the indicated combinations for 48h. Western blotting was used to measure p21 levels (left) while conducting SA-β-gal staining in parallel (middle) with the percentage of senescent cells calculated from the SA-β-gal staining (right).Mammalian two-hybrid assays between pACT-LRP130 and pBIND-PGC1α in A549 cells treated with DMSO or 100nM rapamycin. Samples were subjected to the luciferase activity assays.SIX4 and FOSL2 mRNA (left) and protein levels (right) measured by qPCR and Western blotting, respectively in A549 cells treated with DMSO or 100nM rapamycin. TBP served as a control.qPCR (upper) and Western blotting (lower) assays for GUARDIN, SIX4 and FOSL2 expression in A549 cells comparing shCtrl with shSIX4 (left), shFOSL2 (middle) or FOSL2 (right) after transduction for 48h.Luciferase assays in A549 cells transduced with shCtrl, shSIX4 or shFOSL2 using pGL3 (negative control) or pGL3-FOXO4 promoter reporter plasmids.ChIP assays comparing control IgG versus FOSL2 antibodies demonstrate specific recovery of the GUARDIN promoter by RT-PCR assay. UPe and UPk served as a positive and negative controls, respectively [54].


Figure 1 ( D ) Heatmap showing the expression change of representative NF - κB target genes from the genes with significantly changed expression ( fold change > 2 ) . The data represent the log2 ( gene FPKM ) normalized for each row . RNA - seq data is from 2 independent experiments . ( E ) Intracellular cytokine staining assay of human TNF - α and IL - 2 in sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for 6 hours ( average frequency of positive staining YFP + cells in sh - NC cells without stimulation = 1 ) . ( F ) ELISA analysis of IL - 2 and IL - 6 production in the culture supernatants of human PBMCs transfected with si - NC or si - p38IP , and then stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 ( αCD3 / CD28 ) for 24 hours . ( G and H ) Real - time PCR analysis of TNF - α , IL - 6 and IL - 1β ( G ) and of IFN - β , ISG54 and ISG56 ( H ) mRNA production in RAW264 . 7 cells transfected with si - NC and si - mp38IP - 1 and then stimulated with 500 ng / ml LPS for the indicated times ( average mRNA production in si - NC cells at 0 hour = 1 ) . ( I ) ELISA analysis of TNF - α , IL - 6 , IL - 1β and IFN - β production in the culture supernatants of human PBMCs transfected with si - NC and si - p38IP and then stimulated with 1μg / mL LPS for 24 hours .

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Figure 1(D) Heatmap showing the expression change of representative NF-κB target genes from the genes with significantly changed expression (fold change >2). The data represent the log2 (gene FPKM) normalized for each row. RNA-seq data is from 2 independent experiments.(E) Intracellular cytokine staining assay of human TNF-α and IL-2 in sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for 6 hours (average frequency of positive staining YFP+ cells in sh-NC cells without stimulation = 1).(F) ELISA analysis of IL-2 and IL-6 production in the culture supernatants of human PBMCs transfected with si-NC or si-p38IP, and then stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 (αCD3/CD28) for 24 hours.(G and H) Real-time PCR analysis of TNF-α, IL-6 and IL-1β (G) and of IFN-β, ISG54 and ISG56 (H) mRNA production in RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times (average mRNA production in si-NC cells at 0 hour = 1).(I) ELISA analysis of TNF-α, IL-6, IL-1β and IFN-β production in the culture supernatants of human PBMCs transfected with si-NC and si-p38IP and then stimulated with 1μg/mL LPS for 24 hours.


Figure 2 ( A ) Immunoblot analysis with the indicated antibodies of total cell lysates of sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times . Actin served as a loading control ( throughout ) . ( B ) Statistical analysis of p65 nuclear translocation of sh - NC and sh - p38IP Jurkat E6 . 1 T cells stimulated with 50 ng / ml PMA plus 1µM ionomycin for the indicated times . p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell . * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) . ( C ) Luciferase reporter assay of sh - NC and sh - p38IP Jurkat E6 . 1 cells transfected with NF - κB luc plus an internal control Renilla luciferase reporter for 36 hours , followed by stimulation with ( + ) or without ( - ) 50ng / ml PMA plus 1 µM ionomycin for 8 hours . * * * P < 0 . 001 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) . ( D , E ) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264 . 7 cells transfected with si - NC and si - mp38IP - 1 and then stimulated with 500 ng / ml LPS for the indicated times .

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Figure 2(A) Immunoblot analysis with the indicated antibodies of total cell lysates of sh-NC and sh-p38IP Jurkat E6.1 cells stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times. Actin served as a loading control (throughout).(B) Statistical analysis of p65 nuclear translocation of sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated with 50 ng/ml PMA plus 1µM ionomycin for the indicated times. p65 nuclear translocation was indicated by the fluorescence intensities ratio between the nuclear p65 and the whole cell p65 from each cell. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).(C) Luciferase reporter assay of sh-NC and sh-p38IP Jurkat E6.1 cells transfected with NF-κB luc plus an internal control Renilla luciferase reporter for 36 hours, followed by stimulation with ( + ) or without ( - ) 50ng/ml PMA plus 1 µM ionomycin for 8 hours. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).(D,E) Immunoblot analysis with the indicated antibodies of total cell lysates of RAW264.7 cells transfected with si-NC and si-mp38IP-1 and then stimulated with 500 ng/ml LPS for the indicated times.


Figure 3 ( A ) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography - tandem mass spectrometry . m / z , mass / charge ratio . ( B ) HEK293T cells were transfected with YFP - p38IP , and the cell lysates aliquots were immunoprecipitated with IgG , anti - TAB1 , anti - TAB2 and anti - TAK1 respectively , and then immunoblotted with the indicated antibodies . ( C ) sh - NC or sh - p38IP Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times and lysed , followed byTAK1 immunoprecipitation . TAK1 beads were washed three times with lysis buffer and two times with kinase buffer , then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST - IKKβ ( 111 - 250aa ) protein as substrate to perform in vitro kinase assay , and then immunoblot analysis with p - IKKβ antibody . ( D ) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc - p38IP ( wedge ) , and 24 hours post transfection , the cell lysates were immunoprecipitated with IgG or anti - TAB2 and then immunoblotted with anti - TAK1 and anti - TAB2 . Expression of cMyc - p38IP was determined in the immunoblotted lysates with anti - cMyc . ( E ) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA - p38IP ( wedge ) , and 24 hours post transfection , the cell lysates were immunoprecipitated with IgG or anti - TAK1 and then immunoblotted with anti - TAB2 , anti - TAB1 , anti - HA and anti - TAK1 . Expression of HA - p38IP was determined in the immunoblotted lysates with anti - HA . ( F ) sh - NC and sh - p38IP Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times , and the cell lysates were immunoprecipitated with anti - TAB2 and immunoblotted with anti - TAK1 and anti - TAB2 . ( H ) Jurkat TAg T cells transfected with cMyc vector or cMyc - p38IP were stimulated with 10μg / ml anti - CD3 for the indicated times , and then were lysed and immunoprecipitated with anti - TAK1 antibody and immunoblotted with anti - K63Ub , anti - cMyc and anti - TAK1 antibodies . Lysates were blotted with the indicated antibodies , and actin served as the loading control . ( I ) sh - NC and sh - p38IP Jurkat E6 . 1 T cells stimulated as in ( F ) were lysed and immunoprecipitated with anti - TAK1 and immunoblotted with anti - K63Ub and anti - TAK1 . Lysates were immunoblotted with the indicated antibodies . ( J ) Jurkat TAg T cells transfected and stimulated as in ( H ) were lysed and immunoprecipitated with anti - TAB2 and immunoblotted with anti - K63Ub and anti - TAB2 . ( K ) sh - NC and sh - p38IP Jurkat E6 . 1 cells stimulated as in ( F ) were lysed and immunoprecipitated with anti - TAB2 and immunoblotted with anti - K63Ub and anti - TAB2 . Data ( B - F , H - K ) are representatives 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 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 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 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 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 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 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 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 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

Figure 3(A) Fragmentation spectrum of the p38IP peptide identified in TAK1 complex immunoprecipitated from Jurkat TAg cells by liquid chromatography-tandem mass spectrometry. m/z, mass/charge ratio.(B) HEK293T cells were transfected with YFP-p38IP, and the cell lysates aliquots were immunoprecipitated with IgG, anti-TAB1, anti-TAB2 and anti-TAK1 respectively, and then immunoblotted with the indicated antibodies.(C) sh-NC or sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times and lysed, followed byTAK1 immunoprecipitation. TAK1 beads were washed three times with lysis buffer and two times with kinase buffer, then resuspended in kinase buffer containing 100 μM ATP and 1μg recombinant GST-IKKβ(111-250aa) protein as substrate to perform in vitro kinase assay, and then immunoblot analysis with p-IKKβ antibody.(D) HEK293T cells were transfected with cMyc vector ( - ) or increasing amounts of cMyc-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAB2 and then immunoblotted with anti-TAK1 and anti-TAB2. Expression of cMyc-p38IP was determined in the immunoblotted lysates with anti-cMyc.(E) HEK293T cells were transfected with HA vector ( - ) or increasing amount of HA-p38IP (wedge), and 24 hours post transfection, the cell lysates were immunoprecipitated with IgG or anti-TAK1 and then immunoblotted with anti-TAB2, anti-TAB1, anti-HA and anti-TAK1. Expression of HA-p38IP was determined in the immunoblotted lysates with anti-HA.(F) sh-NC and sh-p38IP Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and the cell lysates were immunoprecipitated with anti-TAB2 and immunoblotted with anti-TAK1 and anti-TAB2.(H) Jurkat TAg T cells transfected with cMyc vector or cMyc-p38IP were stimulated with 10μg/ml anti-CD3 for the indicated times, and then were lysed and immunoprecipitated with anti-TAK1 antibody and immunoblotted with anti-K63Ub, anti-cMyc and anti-TAK1 antibodies. Lysates were blotted with the indicated antibodies, and actin served as the loading control.(I) sh-NC and sh-p38IP Jurkat E6.1 T cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-K63Ub and anti-TAK1. Lysates were immunoblotted with the indicated antibodies.(J) Jurkat TAg T cells transfected and stimulated as in (H) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2.(K) sh-NC and sh-p38IP Jurkat E6.1 cells stimulated as in (F) were lysed and immunoprecipitated with anti-TAB2 and immunoblotted with anti-K63Ub and anti-TAB2. Data (B-F, H-K) are representatives of at least three independent experiments.


Figure 4 ( A ) Jurkat E6 . 1 T cells were stimulated with 10 μg / ml anti - CD3 and 2 μg / ml anti - CD28 for the indicated times , the cell lysates were immunoprecipitated and immunoblotted as indicated . The relative intensity of the IP bands was quantified by densitometry and is shown below the graph . ( B ) HEK293T cells transfected with Flag - tagged TAB2 ( open arrow ) or TAB2 - ub ( solid arrow ) were lysed and immunoprecipitated with anti - Flag , and then immunoblotted with the indicated antibodies . The asterisk indicates a non - specific band . The relative intensity of the IP bands was quantified by densitometry and is presented in the right . ( C ) HEK293T cells co - transfected with HA - tagged p38IP together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) were lysed and immunoprecipitated with anti - cMyc , and then immunoblotted with the indicated antibodies . Asterisk indicates non - specific bands . The relative intensity of the IP bands was quantified by densitometry and is presented in the right . ( D ) HEK293T cells co - transfected with HA - tagged p38IP , Flag - tagged TAB2 - ub , together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) , were lysed and immunoprecipitated with anti - cMyc , and then immunoblotted with the indicated antibodies . The relative intensity of the IP bands was quantified by densitometry and is presented in the right .

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 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 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

Figure 4(A) Jurkat E6.1 T cells were stimulated with 10 μg/ml anti-CD3 and 2 μg/ml anti-CD28 for the indicated times, the cell lysates were immunoprecipitated and immunoblotted as indicated. The relative intensity of the IP bands was quantified by densitometry and is shown below the graph.(B) HEK293T cells transfected with Flag-tagged TAB2 (open arrow) or TAB2-ub (solid arrow) were lysed and immunoprecipitated with anti-Flag, and then immunoblotted with the indicated antibodies. The asterisk indicates a non-specific band. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.(C) HEK293T cells co-transfected with HA-tagged p38IP together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow) were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. Asterisk indicates non-specific bands. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.(D) HEK293T cells co-transfected with HA-tagged p38IP, Flag-tagged TAB2-ub, together with cMyc-tagged TAK1 (open arrow) or ub-TAK1 (solid arrow), were lysed and immunoprecipitated with anti-cMyc, and then immunoblotted with the indicated antibodies. The relative intensity of the IP bands was quantified by densitometry and is presented in the right.


Figure 5 ( A ) GSH - Sepharose beads - immobilized full length or truncated GST - TAK1 proteins were incubated with K63 - linked polyUb chains in 300 μl binding buffer , followed by washing and resolving in SDS - PAGE , and then immunoblotted with anti - ub . GST - fused NEMO serve as positive control and NEMO L329P mutant as negative control . The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST - fused proteins ( lower panel ) . ( C and D ) Immobilized GST - TAB2 was pre - incubated with K63 - linked polyUb chains and then eluted from beads , followed by incubation with eluted GST - TAK1 and GST - p38IP as shown in ( B ) . Incubation mixture were immunoprecipitated with anti - TAB2 or anti - TAK1 , and then immunoblotted with anti - ub , anti - TAB2 , anti - TAK1 and anti - p38IP . ( E ) Statistical analysis of polyUb binding to TAB2 or TAK1 in ( C and D ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) . ( F ) Immobilized GST - TAB2 and GST - TAK1 were respectively pre - incubated with K63 - linked polyUb chains or not as indicated , then eluted from beads , followed by coincubation as indicated . Incubation mixture were immunoprecipitated with anti - TAB2 , and then immunoblotted with anti - TAB2 and anti - TAK1 . The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right . * * * P < 0 . 001 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) . ( G ) Immobilized GST - TAK1 was pre - incubated with K63 - linked polyUb chains or not , then eluted from beads , followed by incubation with eluted GST - p38IP . Incubation mixture were immunoprecipitated with anti - TAK1 , and then immunoblotted with anti - TAK1 and anti - p38IP . The relative intensity of immunoprecipitated p38IP was calculated and shown in the right . * * * P < 0 . 001 ( two - tailed unpaired t - test , mean and s . e . m . ) .

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 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 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 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

Figure 5(A) GSH-Sepharose beads-immobilized full length or truncated GST-TAK1 proteins were incubated with K63-linked polyUb chains in 300 μl binding buffer, followed by washing and resolving in SDS-PAGE, and then immunoblotted with anti-ub. GST-fused NEMO serve as positive control and NEMO L329P mutant as negative control. The blotted membrane was stained with Ponceau S to demonstrate the amount of the input GST-fused proteins (lower panel).(C and D) Immobilized GST-TAB2 was pre-incubated with K63-linked polyUb chains and then eluted from beads, followed by incubation with eluted GST-TAK1 and GST-p38IP as shown in (B). Incubation mixture were immunoprecipitated with anti-TAB2 or anti-TAK1, and then immunoblotted with anti-ub, anti-TAB2, anti-TAK1 and anti-p38IP. (E) Statistical analysis of polyUb binding to TAB2 or TAK1 in (C and D). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).(F) Immobilized GST-TAB2 and GST-TAK1 were respectively pre-incubated with K63-linked polyUb chains or not as indicated, then eluted from beads, followed by coincubation as indicated. Incubation mixture were immunoprecipitated with anti-TAB2, and then immunoblotted with anti-TAB2 and anti-TAK1. The relative intensity of immunoprecipitated TAK1 was calculated and shown in the right. ***P < 0.001; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).(G) Immobilized GST-TAK1 was pre-incubated with K63-linked polyUb chains or not, then eluted from beads, followed by incubation with eluted GST-p38IP. Incubation mixture were immunoprecipitated with anti-TAK1, and then immunoblotted with anti-TAK1 and anti-p38IP. The relative intensity of immunoprecipitated p38IP was calculated and shown in the right. ***P < 0.001 (two-tailed unpaired t-test, mean and s.e.m.).


Figure 6 ( B ) HEK293T cells transfected with N - terminal ( left panels ) or C - terminal ( right panels ) deletion mutants of p38IP as indicated in ( A ) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti - TAK1 , followed by incubation with protein G beads , and then immunoblotted with anti - YFP and anti - TAK1 . ( D ) GST pull down assay . 3μg recombinant GST - T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg / μl , after 4 hrs incubation , washed beads with cell lysis buffer five times and subjected to SDS - PAGE for immunoblotting with anti - TAK1 . ( E ) HEK293T cells transfected with YFP - p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti - TAK1 , followed by incubation with protein G beads , and then immunoblotted with anti - YFP and anti - TAK1 . ( F ) Jurkat TAg cells transfected with YFP vector or YFP - T1B were stimulated with 10μg / ml anti - CD3 for indicated times , then lysed and immunoprecipitated with anti - TAK1 , followed by immunoblotting with the indicated antibodies . ( G ) Primary human PBMCs transfected with YFP vector or YFP - T1Bwere stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 for the indicated times , then lysed and analyzed by immunoblotting . ( H ) Intracellular cytokine staining analysis of TNF - α ( upper ) and IL - 2 ( lower ) in primary human PBMCs transfected and stimulated as in ( F ) for 3 hours ( TNF - α ) and 8 hours ( IL - 2 ) .

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 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 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 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

Figure 6(B) HEK293T cells transfected with N-terminal (left panels) or C-terminal (right panels) deletion mutants of p38IP as indicated in (A) were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.(D) GST pull down assay. 3μg recombinant GST-T1B protein which immobilized on glutathione Sepharose beads was incubated with the cell lysates that obtained by adding 1ml lysis buffer to the HEK293T cells in 10 cm cell culture dish with a final protein concentration around 1μg/μl, after 4 hrs incubation, washed beads with cell lysis buffer five times and subjected to SDS-PAGE for immunoblotting with anti-TAK1.(E) HEK293T cells transfected with YFP-p38IP T1B were lysed after 24 hours transfection and immunoprecipitated with IgG or anti-TAK1, followed by incubation with protein G beads, and then immunoblotted with anti-YFP and anti-TAK1.(F) Jurkat TAg cells transfected with YFP vector or YFP-T1B were stimulated with 10μg/ml anti-CD3 for indicated times, then lysed and immunoprecipitated with anti-TAK1, followed by immunoblotting with the indicated antibodies.(G) Primary human PBMCs transfected with YFP vector or YFP-T1Bwere stimulated with 10 μg/ml anti-CD3 plus 10 μg/ml anti-CD28 for the indicated times, then lysed and analyzed by immunoblotting.(H) Intracellular cytokine staining analysis of TNF-α (upper) and IL-2 (lower) in primary human PBMCs transfected and stimulated as in (F) for 3 hours (TNF-α) and 8 hours (IL-2).


Figure 7 ( A ) HEK293T cells co - transfected with YFP - p38IP and Flag - vector , Flag - USP3 or Flag - USP4 as indicated were lysed and immunoprecipitated with anti - Flag antibody and immunoblotted with indicated antibodies . ( B ) HEK293T cells transfected with Flag - USP4 and YFP - vector or YFP - p38IP as indicated were lysed and immunoprecipitated with anti - IgG or anti - TAK1 and then immunoblotted with anti - Flag and anti - TAK1 . ( C ) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti - TAK1 , followed by in vivo ubiquitination assay of TAK1 as described in methods . ( D ) In vitro deubiquitination assay of TAK1 as described in methods . ( E ) sh - NC and sh - p38IP Jurkat E6 . 1 cells transfected with si - NC , si - p38IP , Flag or Flag - USP4 as indicated were stimulated with anti - CD3 / CD28 for 0 and 15 min at 48 hours post transfection , and then lysed and immunoprecipitated with anti - TAK1 under stringent conditions and immunoblotted with anti - ub . Statistical analysis of p - p38 levels was shown below the blot . * * P < 0 . 01 ; n . s . , not significant ( two - tailed unpaired t - test , mean and s . e . m . ) . ( F ) Jurkat E6 . 1 T cells were stimulated with anti - CD3 / CD28 for the indicated times . The cell lysates were immunoprecipitated with anti - p38IP or anti - TAK1 and then immunoblotted with the indicated antibodies . ( G ) sh - NC and sh - p38IP Jurkat E6 . 1 cells were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for the indicated times , and then lysed and immunoprecipitated with anti - TAK1 and immunoblotted with anti - USP4 and anti - TAK1 . ( H ) Mapping the USP4 binding domain of p38IP .

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

Figure 7(A) HEK293T cells co-transfected with YFP-p38IP and Flag-vector, Flag-USP3 or Flag-USP4 as indicated were lysed and immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(B) HEK293T cells transfected with Flag-USP4 and YFP-vector or YFP-p38IP as indicated were lysed and immunoprecipitated with anti-IgG or anti-TAK1 and then immunoblotted with anti-Flag and anti-TAK1.(C) HEK293T cells transfected with plasmids as indicated were lysed and immunoprecipitated with anti-TAK1, followed by in vivo ubiquitination assay of TAK1 as described in methods.(D) In vitro deubiquitination assay of TAK1 as described in methods.(E) sh-NC and sh-p38IP Jurkat E6.1 cells transfected with si-NC, si-p38IP, Flag or Flag-USP4 as indicated were stimulated with anti-CD3/CD28 for 0 and 15 min at 48 hours post transfection, and then lysed and immunoprecipitated with anti-TAK1 under stringent conditions and immunoblotted with anti-ub. Statistical analysis of p-p38 levels was shown below the blot. **P < 0.01; n.s., not significant (two-tailed unpaired t-test, mean and s.e.m.).(F) Jurkat E6.1 T cells were stimulated with anti-CD3/CD28 for the indicated times. The cell lysates were immunoprecipitated with anti-p38IP or anti-TAK1 and then immunoblotted with the indicated antibodies.(G) sh-NC and sh-p38IP Jurkat E6.1 cells were stimulated with 10 μg/ml anti-CD3 plus 2 μg/ml anti-CD28 for the indicated times, and then lysed and immunoprecipitated with anti-TAK1 and immunoblotted with anti-USP4 and anti-TAK1.(H) Mapping the USP4 binding domain of p38IP.


Figure 1 ( F - K ' ) ( F ' , G ' , H ' , I ' ) are the magnified images of the square region outlined in ( F , G , H , I ) . ( F - K ) are merged images , ( F ' - K ' ) are single channel images of ( F - K ) depicting the presence of NBs ( Mira + ) . Clones ( green ) are outlined by dotted lines . All images in this figure , and all following figures are single confocal sections . ( F - F ' and G - G ' ) No Mira + NBs are recovered in deep sections of control or UAS - dpn clones at 16 hr after clone induction . Quantified in E . ( H - H ' and I - I ' ) At 24 hr after clone induction , Mira + NBs are recovered in UAS - dpn but not control clones . Quantified in E . ( J - J ' and K - K ' ) At 72h after clone induction , numerous Mira + NBs are recovered in UAS - dpn but not in control clones . Quantified in E .

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

Figure 1(F-K') (F', G', H', I') are the magnified images of the square region outlined in (F, G, H, I). (F-K) are merged images, (F'-K') are single channel images of (F-K) depicting the presence of NBs (Mira+). Clones (green) are outlined by dotted lines. All images in this figure, and all following figures are single confocal sections. (F-F' and G-G') No Mira+ NBs are recovered in deep sections of control or UAS-dpn clones at 16 hr after clone induction. Quantified in E. (H-H' and I-I') At 24 hr after clone induction, Mira+ NBs are recovered in UAS-dpn but not control clones. Quantified in E. (J-J'and K-K') At 72h after clone induction, numerous Mira+ NBs are recovered in UAS-dpn but not in control clones. Quantified in E.


Figure 2 ( C - L ' ) Representative images of the deep medulla neuronal layer or NB superficial layer in the larval optic lobe , in which UAS - dpn or control are driven in clones by hs flp ( marked by GFP and outlined ) and stained with the stem cell marker , Miranda ( Mira , magenta ) , and various temporal transcription factors ( tTFs ) ( grey ) . ( C - G ' ) In superficial sections of control clones , Mira + superficial NBs express the tTF : Hth , Ey , Slp , D , Tll in concentric patterns . Quantified in M . ( H - L ' ) In deep sections of the NB clone induced via UAS - dpn , Mira + NBs do not express early tTF Hth or late tTF Tll , but are positive for mid tTFs Ey , Slp and D . Quantified in M . ( M ) Quantification of volume of cells that express a specific tTF as % of total Mira + NB volume within a clone . Hth ( Control n = 5 , m = 31 . 86 ± 3 . 89 , UAS - dpn n = 6 , m = 7 . 779 ± 1 . 212 ) , Ey ( Control n = 7 , m = 26 . 05 ± 5 . 302 , UAS - dpn n = 5 , m = 64 . 08 ± 4 . 936 ) , Slp ( Control n = 7 , m = 34 . 87 ± 2 . 753 , UAS - dpn n = 9 , m = 64 . 72 ± 6 . 045 ) , D ( Control n = 4 , m = 34 . 57 ± 15 . 49 , UAS - dpn n = 10 , m = 45 . 2 ± 6 . 339 ) , Tll ( Control , n = 5 m = 15 . 65 ± 4 . 036 , UAS - dpn n = 6 , m = 3 . 584 , ± 1 . 87 ) .

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 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

Figure 2(C-L') Representative images of the deep medulla neuronal layer or NB superficial layer in the larval optic lobe, in which UAS-dpn or control are driven in clones by hs flp (marked by GFP and outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and various temporal transcription factors (tTFs) (grey). (C-G') In superficial sections of control clones, Mira+ superficial NBs express the tTF: Hth, Ey, Slp, D, Tll in concentric patterns. Quantified in M. (H- L') In deep sections of the NB clone induced via UAS-dpn, Mira+ NBs do not express early tTF Hth or late tTF Tll, but are positive for mid tTFs Ey, Slp and D. Quantified in M.(M) Quantification of volume of cells that express a specific tTF as % of total Mira+ NB volume within a clone. Hth (Control n= 5, m= 31.86 ±3.89, UAS-dpn n= 6, m= 7.779 ±1.212), Ey (Control n= 7, m= 26.05 ±5.302, UAS-dpn n= 5, m= 64.08 ±4.936), Slp (Control n= 7, m= 34.87 ±2.753, UAS-dpn n= 9, m= 64.72 ±6.045), D (Control n= 4, m= 34.57 ±15.49, UAS-dpn n= 10, m= 45.2 ±6.339), Tll (Control, n= 5 m= 15.65 ±4.036, UAS-dpn n= 6, m= 3.584, ±1.87).


Figure 3 ( B ) Quantification of % of Slp + cells in Mira + cells in control and UAS - dpn clones ( expressed as the ratio between Slp + volume and total Mira + volume within a clone ) . 24 hr ( Control n = 7 , m = 14 . 25 ± 2 . 696 , UAS - dpn n = 9 , m = 60 . 66 ± 5 . 384 ) , 48 hr ( Control n = 3 , m = 6 . 096 ± 1 . 509 , UAS - dpn n = 8 , m = 43 . 64 ± 3 . 398 ) , 72 hr ( Control n = 6 , m = 35 . 13 ± 3 . 242 , UAS - dpn n = 9 , m = 64 . 72 ± 6 . 045 ) , 96 hr ( Control n = 6 , m = 29 . 85 ± 4 . 951 , UAS - dpn n = 10 , m = 58 . 2 ± 6 . 603 ) . ( C - F ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which UAS - dpn or control are driven by hs flp ( marked by GFP and outlined ) and dissected and stained with the stem cell marker , Miranda ( Mira , magenta ) , and Sloppy - paired ( Slp , grey ) . At 24h and 48h after clone induction , Mira + NBs express mid tTF Slp compared to superficial NB control , quantified in B . ( C ' ' , D ' ' , E ' ' , F ' ' ) are the magnified images of the square region outlined in ( C ' , D ' , E ' , F ' ) , respectively . ( G - J ' ) At 96h after clone induction , Mira + NBs express mid tTF Slp and do not express late tTF Tll , compared to control , quantified in B and K . ( K ) Quantification of % of Tll + cells in Mira + cells in control and UAS - dpn clones ( expressed as the ratio between Tll + volume and total Mira + volume within a clone ) . Control n = 4 , m = 48 . 34 ± 5 . 956 , UAS - dpn n = 5 , m = 4 . 805 ± 2 . 375 .

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 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 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

Figure 3(B) Quantification of % of Slp+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Slp+ volume and total Mira+ volume within a clone). 24 hr (Control n= 7, m= 14.25 ±2.696, UAS-dpn n= 9, m= 60.66 ±5.384), 48 hr (Control n= 3, m= 6.096 ±1.509, UAS-dpn n= 8, m= 43.64 ±3.398), 72 hr (Control n= 6, m= 35.13 ±3.242, UAS-dpn n= 9, m= 64.72 ±6.045), 96 hr (Control n= 6, m= 29.85 ±4.951, UAS-dpn n= 10, m= 58.2 ± 6.603).(C-F'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn or control are driven by hs flp (marked by GFP and outlined) and dissected and stained with the stem cell marker, Miranda (Mira, magenta), and Sloppy-paired (Slp, grey). At 24h and 48h after clone induction, Mira+ NBs express mid tTF Slp compared to superficial NB control, quantified in B. (C'', D'', E'', F'') are the magnified images of the square region outlined in (C', D', E', F'), respectively.(G-J') At 96h after clone induction, Mira+ NBs express mid tTF Slp and do not express late tTF Tll, compared to control, quantified in B and K. (K) Quantification of % of Tll+ cells in Mira+ cells in control and UAS-dpn clones (expressed as the ratio between Tll+ volume and total Mira+ volume within a clone). Control n= 4, m= 48.34 ±5.956, UAS-dpn n= 5, m= 4.805 ±2.375. D


Figure 4Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe , in which UAS - dpn or control ( UAS - luciferase ) are driven by eyR16F10 - GAL4 and stained with the stem cell marker , Miranda ( Mira , magenta ) , ( B - E ) Mira + NBs are recovered in superficial sections of control under eyR16F10 - GAL4 expression at 6 hr APF and 10 hr APF but not from16 hr APF onwards , quantified in J . ( F - I ) Mira + NBs are recovered in deep sections of UAS - dpn driven by eyR16F10 - GAL4 at 6 hr APF , 10 hr APF , and 16 hr APF but not at 24 hr APF , quantified in J . Arrow in H points to the NBs . ( J ) Quantification of % Mira + cells within eyR16F10 - GAL4 expression domain in control and UAS - dpn ( calculated as the ratio of Mira + cell volume as a percentage of total eyR16F10 - GAL4 domain volume ) . 6 hr APF ( Control , n = 3 , m = 14 . 82 ± 3 . 521 , UAS - dpn , n = 6 , m = 18 . 13 ± 1 . 46 ) , 10 hr APF ( Control n = 4 , m = 11 . 65 ± 1 . 605 , UAS - dpn n = 5 , m = 19 . 11 ± 4 . 609 ) , 16 hr APF ( Control n = 4 , m = 0 . 8897 ± 0 . 239 , UAS - dpn n = 7 , m = 16 . 64 ± 3 . 065 ) , 24 hr APF ( Control n = 4 m = 1 . 174 , ± 0 . 7226 , UAS - dpn n = 6 , m = 0 . 8518 ± 0 . 4849 ) . Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe , in which UAS - dpn or control ( UAS - luciferase ) are driven by ok107 - GAL4 ( outlined ) and stained with the stem cell marker , Miranda ( Mira , magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . ( K - L ' ) More Toy + progeny ( grey ) are present within the ok107 - GAL4 domain ( green , outlined ) in UAS - dpn compared to control . ( O - R ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which heat shock induced UAS - dpn or control ( UAS - luciferase ) clones are stained with the stem cell marker , Mira ( magenta ) , late neuronal marker Tll ( grey ) or glial marker Repo ( grey ) . Arrow points towards the small band of Tll + neurons in control clones . ( O - P ' ) At 72 hr after clone induction , there is less Tll expression within UAS - dpn clones than control clone , quantified in S . ( Q - R ' ) At 72 hr after clone induction , there is less Repo expression within UAS - dpn clones than control clones , quantified in T .

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 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 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

Figure 4Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by eyR16F10-GAL4 and stained with the stem cell marker, Miranda (Mira, magenta), (B-E) Mira+ NBs are recovered in superficial sections of control under eyR16F10-GAL4 expression at 6 hr APF and 10 hr APF but not from16 hr APF onwards, quantified in J. (F-I) Mira+ NBs are recovered in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 6 hr APF, 10 hr APF, and 16 hr APF but not at 24 hr APF, quantified in J. Arrow in H points to the NBs. (J) Quantification of % Mira+ cells within eyR16F10-GAL4 expression domain in control and UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). 6 hr APF (Control, n= 3, m= 14.82 ±3.521, UAS-dpn, n= 6, m= 18.13 ±1.46), 10 hr APF (Control n= 4, m= 11.65 ±1.605, UAS-dpn n= 5, m= 19.11 ±4.609), 16 hr APF (Control n= 4, m= 0.8897 ±0.239, UAS-dpn n= 7, m= 16.64 ±3.065), 24 hr APF (Control n= 4 m= 1.174, ±0.7226, UAS-dpn n= 6, m= 0.8518 ±0.4849).Representative images of the superficial medulla NB layer or deep medulla neuronal layer in the larval or pupal optic lobe, in which UAS-dpn or control (UAS-luciferase) are driven by ok107-GAL4 (outlined) and stained with the stem cell marker, Miranda (Mira, magenta), and mid-temporal progeny marker, Toy-GFP (grey). (K-L') More Toy+ progeny (grey) are present within the ok107-GAL4 domain (green, outlined) in UAS-dpn compared to control.(O-R') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which heat shock induced UAS-dpn or control (UAS-luciferase) clones are stained with the stem cell marker, Mira (magenta), late neuronal marker Tll (grey) or glial marker Repo (grey). Arrow points towards the small band of Tll+ neurons in control clones. (O-P') At 72 hr after clone induction, there is less Tll expression within UAS-dpn clones than control clone, quantified in S. (Q-R') At 72 hr after clone induction, there is less Repo expression within UAS-dpn clones than control clones, quantified in T.


Figure 6Representative images of deep medulla neuronal layer in the larval optic lobe , in which UAS - luc ; UAS - dpn and UAS - D ; UAS - dpn are expressed in clones by hs flp , and stained with the stem cell marker , Miranda ( Mira , magenta ) , and / or Slp , Tll or Repo ( grey ) . ( A - B ' ' ) At 72 hr after clone induction , Mira + NBs within UAS - luc ; UAS - dpn clones express Slp ; Mira + NBs within UAS - D ; UAS - dpn clones do not express Slp , quantified in I . ( C - D ' ) Representative images of deep medulla neuronal layer in the larval optic lobe , in which UAS - dpn and UAS - D ; UAS - dpn are expressed in the ok107 - GAL4 expression domain , and stained with the stem cell marker , Mira ( magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . Fewer Toy + cells were recovered within the ok107 - GAL4 domain ( outlined ) in UAS - D ; UAS - dpn compared to UAS - dpn , quantified in E . ( E ) Quantification of % Toy + cells within ok107 - GAL4 expression domain in UAS - dpn and UAS - D ; UAS - dpn ( calculated as the ratio of Toy + cell volume as a percentage of total ok107 - GAL4 domain volume ) . UAS - dpn n = 9 , m = 22 . 78 ± 4 . 174 , UAS - D ; UAS - dpn n = 5 , m = 14 . 42 ± 2 . 394 . ( J - K ) Quantification of % Tll + or Repo + cells within UAS - dpn clones compared to UAS - D ; UAS - dpn clones ( calculated as the ratio of Tll + or Repo + cell volume as a percentage of total clone volume ) . Tll ( UAS - dpn n = 7 , m = 0 . 7198 ± 0 . 2389 , UAS - D ; UAS - dpn n = 9 , m = 35 . 22 ± 4 . 917 ) , Repo ( UAS - dpn n = 7 m = 1 . 488 ± 0 . 2368 , UAS - D ; UAS - dpn n = 9 , m = 14 . 13 ± 2 . 672 ) . ( L - M ) Mira + NBs are present in deep sections of UAS - dpn driven by eyR16F10 - GAL4 at 126h but are absent in UAS - D ; UAS - dpn , quantified in N . ( N ) Quantification of % Mira cells within eyR16F10 - GAL4 expression domain in UAS - dpn and UAS - D ; UAS - dpn ( calculated as the ratio of Mira + cell volume as a percentage of total eyR16F10 - GAL4 domain volume ) . UAS - dpn n = 6 , m = 18 . 13 ± 1 . 46 , UAS - D ; UAS - dpn n = 8 , m = 1 . 859 ± 0 . 4215 .

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 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 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

Figure 6Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-luc; UAS-dpn and UAS-D; UAS-dpn are expressed in clones by hs flp, and stained with the stem cell marker, Miranda (Mira, magenta), and/or Slp, Tll or Repo (grey). (A-B'') At 72 hr after clone induction, Mira+ NBs within UAS-luc; UAS-dpn clones express Slp; Mira+ NBs within UAS-D; UAS-dpn clones do not express Slp, quantified in I. (C-D') Representative images of deep medulla neuronal layer in the larval optic lobe, in which UAS-dpn and UAS-D; UAS-dpn are expressed in the ok107-GAL4 expression domain, and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer Toy+ cells were recovered within the ok107-GAL4 domain (outlined) in UAS-D; UAS-dpn compared to UAS-dpn, quantified in E. (E) Quantification of % Toy+ cells within ok107-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Toy+ cell volume as a percentage of total ok107-GAL4 domain volume). UAS-dpn n= 9, m= 22.78 ±4.174, UAS-D; UAS-dpn n= 5, m= 14.42 ±2.394.(J-K) Quantification of % Tll+ or Repo+ cells within UAS-dpn clones compared to UAS-D; UAS-dpn clones (calculated as the ratio of Tll+ or Repo+ cell volume as a percentage of total clone volume). Tll (UAS-dpn n= 7, m= 0.7198 ±0.2389, UAS-D; UAS-dpn n= 9, m= 35.22 ±4.917), Repo (UAS-dpn n= 7 m= 1.488 ±0.2368, UAS-D; UAS-dpn n= 9, m= 14.13 ±2.672).(L-M) Mira+ NBs are present in deep sections of UAS-dpn driven by eyR16F10-GAL4 at 126h but are absent in UAS-D; UAS-dpn, quantified in N. (N) Quantification of % Mira cells within eyR16F10-GAL4 expression domain in UAS-dpn and UAS-D; UAS-dpn (calculated as the ratio of Mira+ cell volume as a percentage of total eyR16F10-GAL4 domain volume). UAS-dpn n= 6, m= 18.13 ±1.46, UAS-D; UAS-dpn n= 8, m= 1.859 ±0.4215. D


Figure 7 ( A - C ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - luc ) and UAS - dpn and UAS - D ; UAS - dpn are expressed in hs flp clones ( 72 hr after clone induction ) . Miranda ( Mira , magenta ) and EdU ( grey ) . Despite increased number of Mira + cells in UAS - dpn compared to control , each NB underwent slower cell cycle progression , as indicated by EdU + incorporation . Quantified in E . This slow cell cycle speed was not rescued in UAS - D ; UAS - dpn clones . Quantified with UAS - luc ; UAS - dpn in F . ( G - H ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , where UAS - E2f1 ; UAS - CycE ; UAS - dpn or UAS - dpn are expressed in the ok107 - GAL4 domain ( outlined ) , and stained with the stem cell marker , Mira ( magenta ) , mid - tTF , Slp ( grey ) and late progeny marker , Repo ( Cyan ) . Quantified in I - K . UAS - dpn NBs express Slp and do not create Repo + progeny , whereas UAS - E2f1 ; UAS - CycE ; UAS - dpn NBs do not express Slp and create Repo + progeny .

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 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

Figure 7(A-C'') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc) and UAS-dpn and UAS-D; UAS-dpn are expressed in hs flp clones (72 hr after clone induction). Miranda (Mira, magenta) and EdU (grey). Despite increased number of Mira+ cells in UAS-dpn compared to control, each NB underwent slower cell cycle progression, as indicated by EdU+ incorporation. Quantified in E. This slow cell cycle speed was not rescued in UAS-D; UAS-dpn clones. Quantified with UAS-luc; UAS-dpn in F.(G-H''') Representative images of the deep medulla neuronal layer in the larval optic lobe, where UAS- E2f1; UAS- CycE; UAS-dpn or UAS-dpn are expressed in the ok107-GAL4 domain (outlined), and stained with the stem cell marker, Mira (magenta), mid-tTF, Slp (grey) and late progeny marker, Repo (Cyan). Quantified in I-K. UAS-dpn NBs express Slp and do not create Repo+ progeny, whereas UAS- E2f1; UAS- CycE; UAS-dpn NBs do not express Slp and create Repo+ progeny.


Figure 8 ( B - D ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - luc ) , UAS - NACT , and UAS - D ; UAS - NACT are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Sloppy paired ( Slp , grey ) , Tailless ( Tll , cyan ) . UAS - NACT NBs express Slp and not Tll . UAS - D ; UAS - NACT NBs express Slp and Tll , quantified in E - H . ( J - K ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which UAS - NACT and UAS - NACT ; UAS - dpn are driven by ok107 - GAL4 ( marked by GFP and outlined ) and stained with the stem cell marker , Mira ( magenta ) , and mid - temporal progeny marker , Toy - GFP ( grey ) . Fewer cells express Toy + in UAS - NACT ; UAS - dpn compared to UAS - NACT , quantified in I .

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 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

Figure 8(B-D''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-luc), UAS-NACT, and UAS-D; UAS-NACT are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy paired (Slp, grey), Tailless (Tll, cyan). UAS-NACT NBs express Slp and not Tll. UAS-D; UAS-NACT NBs express Slp and Tll, quantified in E-H.(J-K') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which UAS-NACT and UAS-NACT; UAS-dpn are driven by ok107-GAL4 (marked by GFP and outlined) and stained with the stem cell marker, Mira (magenta), and mid-temporal progeny marker, Toy-GFP (grey). Fewer cells express Toy+ in UAS-NACT; UAS-dpn compared to UAS-NACT, quantified in I.


Figure 9 ( A - C ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - lacz ) , lola - Ri , and UAS - D ; lola - Ri are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Sloppy - paired ( Slp , grey ) , Twin of eyeless ( Toy , cyan ) . lola - Ri induces ectopic Mira + NBs in the deep medulla layers , compared to control , quantified in E . ( H - J ' ' ' ) Representative images of the deep medulla neuronal layer in the larval optic lobe , in which control ( UAS - lacz ) , lola - Ri , and UAS - D ; lola - Ri are driven by eyR16F10 - GAL4 ( outlined ) . Miranda ( Mira , magenta ) , Tailless ( Tll , grey ) , Reversed polarity ( Repo , cyan ) . lola - Ri NBs express less Tll compared to control . There are fewer Tll + and Repo + progeny in the eyR16F10 - GAL4 domain in lola - Ri compared to control . UAS - D ; lola - Ri NBs express more Tll as well as more Tll + and Repo + progeny in eyR16F10 - GAL4 domain compared to lola - Ri alone , quantified in K - L .

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 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

Figure 9(A-C''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Sloppy-paired (Slp, grey), Twin of eyeless (Toy, cyan). lola-Ri induces ectopic Mira+ NBs in the deep medulla layers, compared to control, quantified in E.(H-J''') Representative images of the deep medulla neuronal layer in the larval optic lobe, in which control (UAS-lacz), lola-Ri, and UAS-D; lola-Ri are driven by eyR16F10-GAL4 (outlined). Miranda (Mira, magenta), Tailless (Tll, grey), Reversed polarity (Repo, cyan). lola-Ri NBs express less Tll compared to control. There are fewer Tll+ and Repo+ progeny in the eyR16F10-GAL4 domain in lola-Ri compared to control. UAS-D; lola-Ri NBs express more Tll as well as more Tll+ and Repo+ progeny in eyR16F10-GAL4 domain compared to lola-Ri alone, quantified in K-L.


Figure 1b ) Volcano plot of contrast MPX vs healthy controls ; α < = 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins . d ) Boxplots illustrating key proteins that differ between patients with MPX and controls ( p - values and fdr for corresponding contrast MPX vs Control are provided in brackets ) : TTR ( p - value = 9E - 10 , fdr = 2E - 7 ) , LBP ( p - value = 2E - 4 , fdr = 2E - 3 ) , APOC1 ( p - value = 5E - 8 , fdr = 3E - 6 ) , and C9 ( p - value = 2E - 8 , fdr = 2E - 6 ) . Here , as usual , the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg . e ) Correlation between MPX severity ( NSkin lesions ) and protein expression ( y - axis ) . One MPX patient had an unclear additional skin condition ( not a pure case of MPX ) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome ; however , the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity , the log2 ( 1 + NLesions / 15 ) was used . Here NLesions is the number of lesions . R2 shows squared correlation coefficient . MPX patients are colored orange , control patients green .

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 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 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

Figure 1b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.d) Boxplots illustrating key proteins that differ between patients with MPX and controls (p-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (p-value = 9E-10, fdr = 2E-7), LBP (p-value = 2E-4, fdr = 2E-3), APOC1 (p-value = 5E-8, fdr = 3E-6), and C9 (p-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.e) Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green.


Figure 2a ) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX , COVID - 19 , and controls . b ) Scatterplot of log fold - change ( logFC ) for contrast MPX vs control ( C1 , x - axis ) and logFC for contrast COVID - 19 vs control ( C2 , y - axis ) . Only the central part of the cloud is shown here . Three truncated dots ( APOC1 , CRP , and SAA1 ) are shown in the lower left and upper right corner . Differentially abundant ( Regulated ; ' Reg ' ) proteins are color coded , with the red color corresponding to 37 proteins differentially abundant in both MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) , the orange color corresponding to proteins specifically changed in MPX vs control ( C1 ) only ( 16 proteins ) , and the green color corresponding to proteins responding to both MPX vs control ( C1 ) and MPX vs COVID - 19 ( C3 ) ( 3 proteins ) . There are no intersections between COVID - 19 vs control ( C2 ) and MPX vs COVID - 19 ( C3 ) . The blue color corresponds to proteins responding to MPX vs COVID - 19 ( C3 ) , but not in MPX vs control ( C1 ) ( 11 proteins ) , and the pink color to proteins responding to COVID - 19 vs control ( C2 ) only ( 19 proteins ) . The red dotted line shows a linear regression through the red dots , i . e . proteins differentially abundant in MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Note that orange and pink points have the same direction of regulation in both MPXV vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Only green and blue dots ( except three proteins : ADIPOQ , GPLD1 , and IGHV1 - 2 ) have opposite directions in C1 and in C2 . d ) Differentially regulated proteins of patients with MPX and COVID - 19 ( Volcano Plot ) ; α ≤ 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins . The chosen significance level ensured that fdr for this contrast was below 22 % . e ) Key proteins that differ between patients with MPX and COVID - 19 ( Boxplots ) ( p - values and fdr for corresponding contrast MPX vs COVID - 19 are provided in brackets ) : LCP1 ( p - value = 3E - 4 , fdr = 3E - 2 ) , LDHB ( p - value = 2E - 3 , fdr = 6E - 2 ) , CFHR1 ( p - value = 2E - 4 , fdr = 3E - 2 ) . Here the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg . f ) Top 8 proteins of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) ( top ) or COVID - 19 ( n = 10 ) and MPX ( n = 6 ) cases ( bottom ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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 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 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 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 O O O

Figure 2a) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX, COVID-19, and controls.b) Scatterplot of log fold-change (logFC) for contrast MPX vs control (C1, x-axis) and logFC for contrast COVID-19 vs control (C2, y- axis). Only the central part of the cloud is shown here. Three truncated dots (APOC1, CRP, and SAA1) are shown in the lower left and upper right corner. Differentially abundant (Regulated; 'Reg') proteins are color coded, with the red color corresponding to 37 proteins differentially abundant in both MPX vs control (C1) and COVID-19 vs control (C2), the orange color corresponding to proteins specifically changed in MPX vs control (C1) only (16 proteins), and the green color corresponding to proteins responding to both MPX vs control (C1) and MPX vs COVID-19 (C3) (3 proteins). There are no intersections between COVID-19 vs control (C2) and MPX vs COVID-19 (C3). The blue color corresponds to proteins responding to MPX vs COVID-19 (C3), but not in MPX vs control (C1) (11 proteins), and the pink color to proteins responding to COVID-19 vs control (C2) only (19 proteins). The red dotted line shows a linear regression through the red dots, i.e. proteins differentially abundant in MPX vs control (C1) and COVID-19 vs control (C2). Note that orange and pink points have the same direction of regulation in both MPXV vs control (C1) and COVID-19 vs control (C2). Only green and blue dots (except three proteins: ADIPOQ, GPLD1, and IGHV1-2) have opposite directions in C1 and in C2.d) Differentially regulated proteins of patients with MPX and COVID-19 (Volcano Plot); α ≤ 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins. The chosen significance level ensured that fdr for this contrast was below 22%.e) Key proteins that differ between patients with MPX and COVID-19 (Boxplots) (p-values and fdr for corresponding contrast MPX vs COVID-19 are provided in brackets): LCP1 (p-value = 3E-4, fdr = 3E-2), LDHB (p-value = 2E-3, fdr = 6E-2), CFHR1 (p-value = 2E-4, fdr = 3E-2). Here the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.f) Top 8 proteins of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6) (top) or COVID-19 (n=10) and MPX (n=6) cases (bottom). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


Figure 3b ) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls ( Heatmap ) ; p < 0 . 05 with Mann - Whitney U test with FDR - based multiple testing correction . c ) Key proteins that differ between patients with MPX and controls , and COVID - 19 ( Boxplots ) . Dashed blue lines indicate the lowest detected peptide concentration from calibration curves . Statistics based on Mann - Whitney U test with multiple testing corrections . For boxplots , median is indicated by a solid line , hinges show the 25th and 75th percentiles , whiskers show values that , at maximum , are within 1 . 5 times the interquartile range . d ) Top 15 peptides of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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 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

Figure 3b) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls (Heatmap); p < 0.05 with Mann-Whitney U test with FDR-based multiple testing correction.c) Key proteins that differ between patients with MPX and controls, and COVID-19 (Boxplots). Dashed blue lines indicate the lowest detected peptide concentration from calibration curves. Statistics based on Mann-Whitney U test with multiple testing corrections. For boxplots, median is indicated by a solid line, hinges show the 25th and 75th percentiles, whiskers show values that, at maximum, are within 1.5 times the interquartile range.d) Top 15 peptides of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


Figure 1A PTAR1 protein expression in rat tissues . Proteins extracted from various rat tissues ( 15 μg each ) were analyzed by immunoblotting with antibodies against PTAR1 . B Identification of RabGGTβ as a binding protein of PTAR1 . Anti - FLAG immunoprecipitates from control HeLa S3 cells or FLAG - PTAR1 - expressing HeLa S3 cells were analyzed by SDS - PAGE and silver staining . The 35 kDa protein was identified as RabGGTβ by mass spectrometry . The asterisk denotes a degradation product of FLAG - PTAR1 . C Co - immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol . D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography . Column fractions were analyzed by immunoblotting with antibodies against RabGGTα , PTAR1 , and RabGGTβ . RabGGTβ eluted in two peaks ( peak A and peak B ) . E Co - immunodepletion of PTAR1 and RabGGTβ by anti - PTAR1 IgG . PTAR1 was immunodepleted from the Mono Q peak A fraction using anti - PTAR1 IgG - bound protein A beads , and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ . Normal rabbit IgG and anti - PTAR1 IgG preabsorbed with the antigen peptide were used as controls . F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography . Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ . G Gel filtration chromatography of the recombinant PTAR1 - RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column . Inset shows SDS - PAGE and Coomassie staining analysis of the purified complex .

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 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 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 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 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

Figure 1A PTAR1 protein expression in rat tissues. Proteins extracted from various rat tissues (15 μg each) were analyzed by immunoblotting with antibodies against PTAR1.B Identification of RabGGTβ as a binding protein of PTAR1. Anti-FLAG immunoprecipitates from control HeLa S3 cells or FLAG-PTAR1-expressing HeLa S3 cells were analyzed by SDS-PAGE and silver staining. The 35 kDa protein was identified as RabGGTβ by mass spectrometry. The asterisk denotes a degradation product of FLAG-PTAR1.C Co-immunoprecipitation of endogenous PTAR1 and RabGGTβ from rat brain and thymus cytosol.D Fractionation of rat brain cytosol by Mono Q anion exchange column chromatography. Column fractions were analyzed by immunoblotting with antibodies against RabGGTα, PTAR1, and RabGGTβ. RabGGTβ eluted in two peaks (peak A and peak B).E Co-immunodepletion of PTAR1 and RabGGTβ by anti-PTAR1 IgG. PTAR1 was immunodepleted from the Mono Q peak A fraction using anti-PTAR1 IgG-bound protein A beads, and the supernatant was analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ. Normal rabbit IgG and anti-PTAR1 IgG preabsorbed with the antigen peptide were used as controls.F Fractionation of the Mono Q peak A fraction by Superdex 200 gel filtration chromatography. Column fractions were analyzed by immunoblotting with antibodies against PTAR1 and RabGGTβ.G Gel filtration chromatography of the recombinant PTAR1-RabGGTβ complex purified from Sf9 insect cells on a Superdex 200 column. Inset shows SDS-PAGE and Coomassie staining analysis of the purified complex.


Figure 2C Identification of Ykt6 as a protein biotin - geranylated by GGTase - III . Cytosolic proteins extracted from statin - treated HeLa S3 cells were applied to GST or GST - GGTase - III affinity columns . Bound proteins were eluted and incubated with recombinant GGTase - III and BGPP . Reaction products were separated by SDS - PAGE , transferred to a nitrocellulose membrane , and probed with horseradish peroxidase ( HRP ) - labeled avidin to detect biotinylated proteins . Mass spectrometry identified the 25 kDa protein as Ykt6 . D Biotin - geranylation of recombinant Ykt6 by GGTase - III . Bacterially produced recombinant untagged Ykt6 ( left ) was incubated with buffer , GGTase - III , or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC . Biotin - geranylated Ykt6 was

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

Figure 2C Identification of Ykt6 as a protein biotin-geranylated by GGTase-III. Cytosolic proteins extracted from statin-treated HeLa S3 cells were applied to GST or GST-GGTase-III affinity columns. Bound proteins were eluted and incubated with recombinant GGTase-III and BGPP. Reaction products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase (HRP)-labeled avidin to detect biotinylated proteins. Mass spectrometry identified the 25 kDa protein as Ykt6.D Biotin-geranylation of recombinant Ykt6 by GGTase-III. Bacterially produced recombinant untagged Ykt6 (left) was incubated with buffer, GGTase-III, or RabGGTase in the absence or presence of BGPP for 30 min at 37ºC. Biotin-geranylated Ykt6 was detected


Figure 3C Farnesylation of recombinant Ykt6 by FTase . Recombinant Ykt6 ( 5 μM ) was incubated with FTase ( 0 or 50 nM ) and 3H - FPP ( 1 μM ; ~ 3 , 500 dpm / pmol ) at 37ºC . Reactions were stopped at the indicated time points , and the amount of 3H - farnesyl transferred to Ykt6 was quantified by scintillation counting ( mean ± SEM , n = 3 ) . D Geranylgeranylation of recombinant Ykt6 by GGTase - III . Recombinant Ykt6 ( 5 μM ) was incubated with increasing concentrations of GGTase - III ( 0 - 100 nM ) and 3H - GGPP ( 1 μM ; ~ 3 , 000 dpm / pmol ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) . E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase - III . WT and mutant Ykt6 proteins ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified ( mean ± SEM , n = 3 ) . G Mass spectra of prenylated Ykt6 peptides . Recombinant Ykt6 was farnesylated by FTase ( FTase ) , further processed by RCE1 and ICMT ( RCE1 / ICMT ) , and finally geranylgeranylated by GGTase - III ( GGTase - III ) . After purification to homogeneity , the prenylated Ykt6 proteins were digested with chymotrypsin ( left ) or V8 protease ( right ) and analyzed by MALDI - TOF mass spectrometry . The calculated m / z values for the C - terminal peptide ions are shown in parentheses . The peaks labeled with an asterisk correspond to amino acids 14 - 38 of Ykt6 ( AKVVLLKAAYDVSSFSFFQRSSVQE ; m / z 2806 . 48 ) . Effects of C - terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase - III . Purified recombinant unprenyl Ykt6 , Cys195 - farnesyl Ykt6 , and Cys195 - farnesyl Ykt6ΔAIM ( 5 μM each ) were incubated with GGTase - III ( 100 nM ) and 3H - GGPP ( 1 μM ) for the indicated times , and the amount of 3H - geranylgeranyl transferred to Ykt6 was quantified . ( mean ± SEM , n = 3 ) . I Gel filtration chromatography of purified unprenyl Ykt6 , Cys195 - farnesyl Ykt6ΔAIM , and Cys194 / 195 - diprenyl Ykt6ΔAIM ( 135 μg each ) on a Superdex 200 column . Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison . The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6 , suggesting more compact and stable folds .

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 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 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 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

Figure 3C Farnesylation of recombinant Ykt6 by FTase. Recombinant Ykt6 (5 μM) was incubated with FTase (0 or 50 nM) and 3H-FPP (1 μM; ~3,500 dpm/pmol) at 37ºC. Reactions were stopped at the indicated time points, and the amount of 3H-farnesyl transferred to Ykt6 was quantified by scintillation counting (mean ± SEM, n = 3).D Geranylgeranylation of recombinant Ykt6 by GGTase-III. Recombinant Ykt6 (5 μM) was incubated with increasing concentrations of GGTase-III (0-100 nM) and 3H-GGPP (1 μM; ~3,000 dpm/pmol) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).E Geranylgeranylation of Cys to Ser mutants of Ykt6 by GGTase-III. WT and mutant Ykt6 proteins (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified (mean ± SEM, n = 3).G Mass spectra of prenylated Ykt6 peptides. Recombinant Ykt6 was farnesylated by FTase (FTase), further processed by RCE1 and ICMT (RCE1/ICMT), and finally geranylgeranylated by GGTase-III (GGTase-III). After purification to homogeneity, the prenylated Ykt6 proteins were digested with chymotrypsin (left) or V8 protease (right) and analyzed by MALDI-TOF mass spectrometry. The calculated m/z values for the C-terminal peptide ions are shown in parentheses. The peaks labeled with an asterisk correspond to amino acids 14-38 of Ykt6 (AKVVLLKAAYDVSSFSFFQRSSVQE; m/z 2806.48).Effects of C-terminal processing and Cys195 farnesylation on Cys194 geranylgeranylation by GGTase-III. Purified recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, and Cys195-farnesyl Ykt6ΔAIM (5 μM each) were incubated with GGTase-III (100 nM) and 3H-GGPP (1 μM) for the indicated times, and the amount of 3H-geranylgeranyl transferred to Ykt6 was quantified. (mean ± SEM, n = 3).I Gel filtration chromatography of purified unprenyl Ykt6, Cys195-farnesyl Ykt6ΔAIM, and Cys194/195-diprenyl Ykt6ΔAIM (135 μg each) on a Superdex 200 column. Dotted lines indicate the elution profile of unprenyl Ykt6 for comparison. The prenylated Ykt6 proteins were eluted slightly later than the unprenylated Ykt6, suggesting more compact and stable folds.