Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 2 - month - , 12 - month - , 2 - month - old mice after 4 days of challenge of C57BL / 6 Cx3cr1GFP / GFP mouse RPE / choroid plexus ( aging : n = 5 - 6 / group ; Mann - Whitney U - : 12 months * P = 0 . 0129 ; 4 days challenge : n = 7 - 8 / group ; * P = 0 . 029 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD B-ORGANISM B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 2-month-, 12-month-, and 2-month-old mice after 4 days of light challenge of C57BL/6 and Cx3cr1GFP/GFP mouse RPE/choroid plexus (aging: n = 5-6/group; Mann-Whitney U-test: 12 months *P = 0.0129; 4 days light challenge: n = 7-8/group; *P = 0.029).


Immunohistochemistry of FASL ( red ) IBA - 1 ( green ) of a RPE flatmounts sections ( insets , Hoechst blue ) from a 12 - month - old WT ( B ) Cx3cr1GFP / GFP ( C ) mouse ( representative of three independent experiments , immunostainings omitting antibody served as negative controls ) .

B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O

Immunohistochemistry of FASL (red) and IBA-1 (green) of a RPE flatmounts and sections (insets, Hoechst staining blue) from a 12-month-old WT (B) and Cx3cr1GFP/GFP(C) mouse (representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus extracts 3 h after subretinal injection of C57BL / 6J macrophages or Cx3cr1GFP / GFP macrophages ( n = 6 per group ) , Mann - Whitney U - , * P = 0 . 0087 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus extracts 3 h after subretinal injection of C57BL/6J macrophages or Cx3cr1GFP/GFP macrophages (n = 6 per group), Mann-Whitney U-test, *P = 0.0087.


IBA - 1 - stained RPE flatmounts of 2 - month - old C57BL / 6J , Faslgld / gld , Faslpr / lpr mice after 4 days of challenge . of subretinal IBA - 1 + mononuclear phagocytes in control ( left ) 4 - day - challenged ( right ) 2 - month - old mice of indicated strains ( n = 6 - 10 / group ANOVA / Dunnett at 4 - day challenge : C57BL / 6J versus FasLgld / gld C57BL / 6J versus Faslpr / lpr both * P < 0 . 0001 ; Mann - Whitney U - at 4 - day challenge : C57BL / 6J versus FasLgld / gld * P < 0 . 0001 ; C57BL / 6J versus Faslpr / lpr * P < 0 . 0001 ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O

IBA-1-stained RPE flatmounts of 2-month-old C57BL/6J, Faslgld/gld, and Faslpr/lpr mice after 4 days of light challenge.Quantification of subretinal IBA-1+ mononuclear phagocytes in control (left) and 4-day light-challenged (right) 2-month-old mice of the indicated strains (n = 6-10/group ANOVA/Dunnett test at 4-day light challenge: C57BL/6J versus FasLgld/gld and C57BL/6J versus Faslpr/lpr both *P < 0.0001; Mann-Whitney U-test at 4-day light challenge: C57BL/6J versus FasLgld/gld *P < 0.0001; C57BL/6J versus Faslpr/lpr *P < 0.0001).


of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injection of C57BL / 6JCFSE + macrophages into C57BL / 6J FasLgld / gldmice Faslpr / lprmacrophages into C57BL / 6Jmice ( n = 10 - 15 / group , one - way ANOVA / Dunnett : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into Fasgld / gldmice * P = 0 . 0002 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 . Mann - Whitney U - : C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus C57BL / 6Jmacrophages inj . into FasLgld / gldmice * P < 0 . 0001 ; C57BL / 6Jmacrophages inj . into C57BL / 6Jmice versus Faslpr / lprmacrophages inj . into C57BL / 6Jmice * P < 0 . 0001 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O

Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6JCFSE+macrophages into C57BL/6J and FasLgld/gldmice and Faslpr/lprmacrophages into C57BL/6Jmice (n = 10-15/group, one-way ANOVA/Dunnett test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into Fasgld/gldmice *P = 0.0002; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001. Mann-Whitney U-test: C57BL/6Jmacrophages inj. into C57BL/6Jmice versus C57BL/6Jmacrophages inj. into FasLgld/gldmice *P < 0.0001; C57BL/6Jmacrophages inj. into C57BL/6Jmice versus Faslpr/lprmacrophages inj. into C57BL/6Jmice *P < 0.0001).


of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFP CFSE + macrophages into C57BL / 6J with or without Fas agonist MegaFasL ( calculated intraocular concentrations 1 ng / ml ; n = 7 - 8 ; Mann - Whitney U - : * P = 0 . 014 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFP CFSE+ macrophages into C57BL/6J with or without the Fas agonist MegaFasL (calculated intraocular concentrations 1 ng/ml; n = 7-8; Mann-Whitney U-test: *P = 0.014).


In vitro MegaFasL - induced apoptosis of monocytes macrophages of indicated genotypes cultured for 24 h . TUNEL + nucleus expressed as of non - MegaFasL - exposed control .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O

In vitro MegaFasL-induced apoptosis of monocytes and macrophages of the indicated genotypes cultured for 24 h. TUNEL+ nucleus quantification expressed as percentage of non-MegaFasL-exposed control.


Quantitative RT - PCR of FasLmRNA normalized with β - actinmRNA of 12 - month - old C57BL / 6 RPE / choroid plexus 3 h after subretinal injection of PBS ( PBS ; n = 36 ) , IL - 6 ( n = 17 ) , APOE3 ( n = 21 ) ; calculated intraocular concentrations : 5 ng / ml 10 μg / ml , respectively ; one - way ANOVA / Dunnett post hoc PBS versus IL - 6 * P = 0 . 038 ; t - PBS versus IL - 6 * P = 0 . 043 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

Quantitative RT-PCR of FasLmRNA normalized with β-actinmRNA of 12-month-old C57BL/6 RPE/choroid plexus 3 h after subretinal injection of PBS (PBS; n = 36), IL-6 (n = 17), and APOE3 (n = 21); calculated intraocular concentrations: 5 ng/ml and 10 μg/ml, respectively; one-way ANOVA/Dunnett post hoc test PBS versus IL-6 *P = 0.038; t-test PBS versus IL-6 *P = 0.043.


Mouse IL - 6 ELISA of supernatants from C57BL / 6J peritoneal macrophages incubated for 24 h in control medium , lipid - free APOE3 ( 5 μg / ml ) , APOE3 ( 5 μg / ml ) , polymyxin B ( 25 μg / ml ) , heat - denatured APOE3 ( dAPOE3 , 5 μg / ml ) , APOE3 ( 5 μg / ml ) , rat IgG1 isotype control ( IgG , 100 μg / ml ) or APOE3 ( 5 μg / ml ) rat anti - CD14 antibody ( aCD14 Ab , 100 μg / ml ) or APOE3 ( 5 μg / ml ) rat anti - TLR2 antibody ( aTLR2 Ab , 100 μg / ml ) ( n = 5 - 6 / group ; one - way ANOVA / Bonferroni multi - comparison : APOE3 versus CTL * P < 0 . 0001 ; dAPOE3 versus APOE3 # P < 0 . 0001 ; APOE3 IgG versus CTL P < 0 . 0001 ; APOE3 IgG versus APOE3 aCD14 Ab § P < 0 . 0001 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P < 0 . 0001 . Mann - Whitney U - : APOE3 versus CTL * P = 0 . 0043 ; dAPOE3 versus APOE3 # P = 0 . 0117 ; APOE3 IgG versus CTL P = 0 . 0080 ; APOE3 IgG versus APOE3 aCD14 Ab § P = 0 . 0117 ; APOE3 IgG versus APOE3 aTLR2 Ab + + P = 0 . 0079 . experiment was repeated twice with similar results ) .

B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O

Mouse IL-6 ELISA of supernatants from C57BL/6J peritoneal macrophages incubated for 24 h in control medium, lipid-free APOE3 (5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), heat-denatured APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype control (IgG, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-CD14 antibody (aCD14 Ab, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-TLR2 antibody (aTLR2 Ab, 100 μg/ml) (n = 5-6/group; one-way ANOVA/Bonferroni multi-comparison tests: APOE3 versus CTL *P < 0.0001; dAPOE3 versus APOE3 #P < 0.0001; APOE3 IgG versus CTL P < 0.0001; APOE3 IgG versus APOE3 aCD14 Ab §P < 0.0001; APOE3 IgG versus APOE3 aTLR2 Ab ++P <0.0001. Mann-Whitney U-test: APOE3 versus CTL *P = 0.0043; dAPOE3 versus APOE3 #P = 0.0117; APOE3 IgG versus CTL P = 0.0080; APOE3 IgG versus APOE3 aCD14 Ab §P = 0.0117; APOE3 IgG versus APOE3 aTLR2 Ab ++P = 0.0079. The experiment was repeated twice with similar results).


Quantitative RT - PCR of IL - 6mRNA normalized with S26mRNA of C57BL / 6J Cx3cr1GFP / GFP , Cx3cr1GFP / GFP ApoE − / − peritoneal macrophages cultured for 24 h with CX3CL1 ( n = 5 per group , one - way ANOVA / Bonferroni post hoc multi - comparison : C57BL / 6J versus Cx3cr1GFP / GFP * P < 0 . 0001 Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P < 0 . 0001 . Mann - Whitney U - : C57BL / 6J versus Cx3cr1GFP / GFP * P = 0 . 0079 Cx3cr1GFP / GFP versus Cx3cr1GFP / GFPApoE − / − ‡ P = 0 . 0079 . experiment was repeated twice with similar results . ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR of IL-6mRNA normalized with S26mRNA of C57BL/6J and Cx3cr1GFP/GFP, and Cx3cr1GFP/GFP ApoE−/− peritoneal macrophages cultured for 24 h with CX3CL1 (n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1GFP/GFP *P < 0.0001 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P < 0.0001. Mann-Whitney U-test: C57BL/6J versus Cx3cr1GFP/GFP *P = 0.0079 and Cx3cr1GFP/GFP versus Cx3cr1GFP/GFPApoE−/−‡P = 0.0079. The experiment was repeated twice with similar results.).


Orthogonal lateral Z - stack projection of IL - 6 ( D , red ) , IBA - 1 + ( E , green ) , phalloidin ( E , blue ) of IBA + mononuclear phagocytes adjacent to phalloidin + RPE of a retinal flatmount from a 12 - month - old Cx3cr1GFP / GFP mouse . ( Representative of three independent experiments , immunostainings omitting antibody served as negative controls ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY O B-TISSUE O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O O O O O O

Orthogonal and lateral Z-stack projection of IL-6 (D, red), IBA-1+ (E, green), and phalloidin (E, blue) of IBA+ mononuclear phagocytes adjacent to the phalloidin+ RPE of a retinal flatmount from a 12-month-old Cx3cr1GFP/GFP mouse. (Representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls).


of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injection of C57BL / 6J CFSE + macrophages into C57BL / 6J with or without IL - 6 ( n = 15 - 20 / group ; calculated intraocular concentrations of 5 ng / ml . Mann - Whitney U - : * P < 0 . 0001 ) . of subretinal CFSE + F4 / 80 + macrophages on RPE retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP / GFPCFSE + Mϕs into C57BL / 6J with control ( IgG , n = 16 ) or anti - IL - 6 antibody ( aIL - 6 Ab , n = 8 ; calculated intraocular concentrations 5 μg / ml ; per group . Mann - Whitney U - : * P = 0 . 0036 ) .

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of subretinal CFSE+F4/80+ macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6J CFSE+ macrophages into C57BL/6J with or without IL-6 (n = 15-20/group; calculated intraocular concentrations of 5 ng/ml. Mann-Whitney U-test: *P < 0.0001).Quantification of subretinal CFSE+F4/80+macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1GFP/GFPCFSE+Mϕs into C57BL/6J with control (IgG, n = 16) or anti-IL-6 antibody (aIL-6 Ab, n = 8; calculated intraocular concentrations 5 μg/ml; per group. Mann-Whitney U-test: *P = 0.0036).


7 day - injured IBA - 1 ( green ) CD102 ( red ) double - stained RPE flatmounts of control IgG - ( I ) anti - IL - 6 - treated ( J ) Cx3cr1GFP / GFP mice . of subretinal IBA - 1 + mononuclear phagocytes / impact localized on lesion surrounding RPE of Cx3cr1GFP / GFP mice treated with control IgG , IL - 6 - , or CD14 - blocking antibodies ( calculated intraocular 5 μg / ml ; n = 13 - 14 / group , one - way ANOVA / Dunnett ' s post hoc of IgG versus any other group * P < 0 . 001 . Mann - Whitney U - IgG versus anti - IL - 6 * P = 0 . 0021 ; IgG versus anti CD14 * P = 0 . 0028 ) .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-TISSUE O O O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O B-TISSUE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O

7 day laser-injured IBA-1 (green) and CD102 (red) double-stained RPE flatmounts of control IgG- (I) and anti-IL-6-treated (J) Cx3cr1GFP/GFP mice.Quantification of subretinal IBA-1+ mononuclear phagocytes/impact localized on the lesion surrounding RPE of Cx3cr1GFP/GFP mice treated with control IgG, IL-6-, or CD14-blocking antibodies (calculated intraocular concentration 5 μg/ml; n = 13-14/group, one-way ANOVA/Dunnett's post hoc tests of IgG versus any other group *P < 0.001. Mann-Whitney U-test IgG versus anti-IL-6 *P = 0.0021; IgG versus anti CD14*P = 0.0028).


heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . data for three sensing channels are reported as an average response . injections are delimited by red arrows . A Injection of 100 nM FGF2 .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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-GENEPROD O

heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. A Injection of 100 nM FGF2.


heparin . Reducing - end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface . PBS running buffer flow rate was 500 µL . min - 1 . data for three sensing channels are reported as an average response . injections are delimited by red arrows . B 64 nM S1 RBD . C 64 nM S1 RBD over a streptavidin surface ( no heparin functionalization ) .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O

heparin.Reducing-end biotinylated heparin was immobilised on a streptavidin functionalized P4SPR sensor surface. PBS running buffer flow rate was 500 µL.min-1. The data for the three sensing channels are reported as an average response. Protein injections are delimited by the red arrows. B 64 nM S1 RBD protein. C 64 nM S1 RBD protein over a streptavidin surface (no heparin functionalization).


structural change of SARS - CoV - 2 S1 RBD observed in presence of heparin by circular dichroism ( CD ) spectroscopy . ( A ) CD spectra of 200 μg / mL spike 1 alone ( purple ) or with 30 μg heparin ( orange ) in phosphate buffered saline pH 7 . 4 . Theoretical sum of spike 1 alone 30 μg of heparin ( control ) if no interaction was observed ( black ) . ( B ) Δ secondary structure ( % ) of ( A ) . A 2 . 4 % increase in helix 2 . 2 % decrease in antiparallel secondary structural features were calculated ( BestSel ) for observed spectrum compared to that of theoretical , summative spectrum of SARS - CoV - 2 S1 RBD in presence of heparin .

O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O

The structural change of the SARS-CoV-2 S1 RBD observed in the presence of heparin by circular dichroism (CD) spectroscopy.(A) CD spectra of 200 μg/mL spike 1 alone (purple) or with 30 μg heparin (orange) in phosphate buffered saline pH 7.4. Theoretical sum of spike 1 alone and 30 μg of heparin (control) if no interaction was observed (black). (B) Δ secondary structure (%) of (A). A 2.4% increase in helix and 2.2% decrease in antiparallel secondary structural features were calculated (BestSel) for the observed spectrum compared to that of the theoretical, summative spectrum of the SARS-CoV-2 S1 RBD in the presence of heparin.


D - G Culture dynamics of phage ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . strains were exposed to phage at time = 0 h , optical density measurements were read in a 96 ‐ well plate format . Each experiment was performed three times with three technical triplicates for each biological replicate . Error bars represent . Shown are representative results for three of ten phages tested ; data for remaining seven phages are found in Supplementary Fig S2 .

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D-G Culture dynamics of phage‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Bacterial strains were exposed to phage at time = 0 h, and optical density measurements were read in a 96‐well plate format. Each experiment was performed three times with three technical triplicates for each biological replicate. Error bars represent SEM. Shown are representative results for three of the ten phages tested; data for the remaining seven phages are found in Supplementary Fig S2.


A Culture dynamics over an extended period ( > 30 h ) for SPO1 ‐ infected wild ‐ type ( black ) versus BREX ‐ containing ( red ) strains of B . subtilis BEST7003 . Each curve represents a single technical replicate grown in a single well on a 96 ‐ well plate . Culture decline is temporally reproducible for BREX ‐ lacking but occurs later , at apparently stochastic time points , for BREX ‐ containing . Re ‐ growth following culture crash represents phage ‐ resistant mutants .

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A Culture dynamics over an extended period (> 30 h) for SPO1‐infected wild‐type (black) versus BREX‐containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96‐well plate. Culture decline is temporally reproducible for the BREX‐lacking strain but occurs later, at apparently stochastic time points, for the BREX‐containing strain. Re‐growth following culture crash represents phage‐resistant mutants.


B Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with SPO1 . Error bars represent SD . Y ‐ axis represents absolute phage concentrations . Black red arrows point to time point of maximal burst for BREX ‐ lacking BREX ‐ containing strains , respectively .

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B Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y‐axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX‐lacking and BREX‐containing strains, respectively.


C Phage production during a one ‐ step phage growth curve experiment with wild ‐ type ( black ) BREX ‐ containing ( red ) strains of B . subtilis BEST7003 infected with Φ3T . Error bars represent SD . Y ‐ axis represents relative phage concentrations normalized to value at beginning of infection .

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C Phage production during a one‐step phage growth curve experiment with wild‐type (black) and BREX‐containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection.


D Multiplex PCR showing lysogeny during a phage infection time course in lacking BREX ( black ) , but not in BREX ‐ containing ( red ) , or uninfected ( U ) strains . Amplicons for , phage , lysogen ‐ specific are 293 , 485 , 1 , 218 bp , respectively .

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D Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX‐containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen‐specific DNA are 293, 485, and 1,218 bp, respectively.


A BREX does not work via an abortive infection mechanism . Increasing MOI of Φ3T infection from 0 . 05 to 5 shortens time to culture crash for BREX ‐ lacking , but does not result in culture decline for BREX ‐ containing . Error bars represent SD of technical triplicates .

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A BREX does not work via an abortive infection mechanism. Increasing the MOI of the Φ3T infection from 0.05 to 5 shortens the time to culture crash for the BREX‐lacking strain, but does not result in culture decline for BREX‐containing strain. Error bars represent SD of technical triplicates.


B system does not interfere with phage adsorption to . Strains either containing ( red ) or lacking ( black ) BREX system were infected with phage Φ3T then chloroform ‐ treated 15 min following infection . culture was plated on Φ3T ‐ sensitive B . subtilis plaques , representing extracellular , unadsorbed phages , were counted .

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B The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform‐treated 15 min following infection. The culture was plated on Φ3T‐sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted.


C Phage replication does not occur in BREX ‐ containing ( red ) , but is observed in BREX ‐ lacking ( black ) . Y ‐ axis represents relative phage concentrations normalized to value at beginning of infection , as measured by Illumina sequencing .

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C Phage DNA replication does not occur in BREX‐containing cells (red), but is observed in the BREX‐lacking strain (black). Y‐axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing.


C , D Culture dynamics of non ‐ infected ( C ) or phage ‐ infected ( D ) cultures . Curves depict culture dynamics of strains lacking BREX ( black ) BREX ‐ containing ( red ) strains of B . subtilis BEST7003 , as well as a BREX ‐ containing where pglX methylase was deleted ( green ) . Axes error bars are as in Fig .

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C, D Culture dynamics of non‐infected (C) or phage‐infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX‐containing (red) strains of B. subtilis BEST7003, as well as a BREX‐containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig .


E Southern blot of phage Φ3T genome during infection . Numbers indicate time ( in min ) following infection ; U , uninfected . Probe was designed to match positions 94 , 645 - 95 , 416 in phage genome . Each lane contains 200 ng total .

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E Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645-95,416 in the phage genome. Each lane contains 200 ng total DNA.


( B - D ) of normalized bouton number ( ( B ) , total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) , normalized axon ( C ) normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * Data information : In ( B - D bars are labelled with number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction. (n.s. = not significant; p<0.05 = *; p<0.001= ***; p<0.0001=**** Data information: In (B-D bars are labelled with the number of replicates.


( B , C ) Western Blot showing enrichments upon pulldown of same probes as in ( A ) incubated with increasing concentrations of purified recombinant GST - Ythdf ( B ) His - NT - Fmr1 ( C ) . of m6A / A signal is shown below blot as median + / - of triplicates . Both bind more efficiently upon methylation .

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(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.


( D ) Results of polarization ( FP ) using Cy5 labelled probes containing four AAACU m6A consensus sites , with or without methylation , incubated with GST - Ythdf His - NT - Fmr1 . relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8 . Binding constants ( kd values ) were determined by fitting a Michaelis - Menten non - linear regression onto relative FP values in GraphPad Prism 8 , if applicable . respective binding constants are given in table next to graphs .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(D) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.


( B - D ) of normalized bouton number ( ( B ) , total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) , normalized axon ( C ) normalized branching ( D ) of NMJ 6 / 7 in A2 - A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ; p < 0 . 001 = * * * ; p < 0 . 0001 = * * * * ) . Data information : In ( B - D , , bars are labelled with number of replicates .

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(B-D) Quantification of normalized bouton number ((B), total number of boutons/muscle surface area (μm2 × 1,000)), normalized axon length (C) and normalized branching (D) of NMJ 6/7 in A2-A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.05 = *; p<0.01 = **; p<0.001= ***; p<0.0001=****). Data information: In (B-D, , bars are labelled with the number of replicates.


( B ) Co - immunoprecipitation experiment in S2R + co - expressing FlagMyc - tagged Ythdf Myc - tagged Fmr1 . Ythdf was used as a bait via its Flag tag . lysate was treated with Benzonase as indicated to remove interactions enabled by .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O

(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.


Pulldown of biotinylated probes of ( A ) repetitive GGACU sequences incubated with 4 pmol His - NT - Fmr1 increasing concentrations of GST - Ythdf indicated P - values were determined by unpaired , two - Student ' s t - .

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Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf The indicated P-values were determined by unpaired, two-tailed Student's t-test.


( A ) Representative Western blot of extracts of late pupae heads ( 85 - 95h ) from control , Ythdf # 0 / Ythdf # 0 Fmr1 ∆ 50 / Fmr1 ∆ 50 flies . membranes were probed with anti - Profilin or anti - Actin antibodies . ( B ) of Profilin ( mean ± SD ) obtained from three independent - extraction Western blot as in ( A ) . was performed using Fiji . p < 0 . 005 = * * ; p < 0 . 05 = * , measured with unpaired t - .

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(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies. (B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.


( D ) of futsch mRNA obtained from isolated 3rd instar larvae brains of indicated genotypes via real - time PCR . Bars show average ± SD of biological triplicates . Bars are labelled with number of replicates .

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(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates. Bars are labelled with the number of replicates.


( F ) of normalized bouton number ( total number of boutons / muscle surface ( μm2 × 1 , 000 ) ) of NMJ 6 / 7 in hemisegment A3 of indicated genotypes . Bars show mean ± s . e . m . Multiple comparisons were performed using one - way ANOVA with a post - hoc Sidak - Bonferroni correction ( n . s . = not significant ; p < 0 . 01 = * * ; p < 0 . 0001 = * * * * ) . Bars are labelled with number of replicates .

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(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****). Bars are labelled with the number of replicates.


E - G ) Representative Western blot of level ( E ) of translation efficiency of S2R + transfected with wildtype or mutated futsch reporter constructs upon control or Mettl3 KD ( F ) or Ythdf KD ( G ) . LacZ dsRNA was used for control KD . translation efficiency was calculated as of relative GFP level GFP mRNA level . mCherry served as a normalization control for both mRNA level . Bars show mean ± s . e . m of biological triplicates . P values were determined with a Student ' s t - . ( n . s . = not significant ; p < 0 . 05 = * ; p < 0 . 01 = * * ) .

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E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).


A Transcriptome re - of ulp2Δ strains compared with WT ( MHY1379 ) from previous - seq experiments Two - dimensional agglomerative hierarchical clustering shows 2 , 903 significantly up - or down - regulated genes ( P < 0 . 05 ) in triplicate samples . Red blue indicate up down regulation of genes , respectively , their represents relative gene expression changes . B Summery of transcriptome data in ( A ) . gray colors indicate up - down - regulated genes , respectively , in ulp2Δ . pie graph shows percentages of significantly changed genes , except for 130 ribosomal genes ( RPs ) , are classified by FPKM ( Fragments Per Kilobase of Million reads mapped ) values in bar graph .

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A Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up and down regulation of genes, respectively, and their intensity represents the relative gene expression changes. B Summery of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph.


C qRT - PCR of highly transcribed PMA1 , ADH1 PYK1 genes in ulp2Δ . Expression was measured relative to WT data were normalized to SPT15 expression . Error bars indicate standard deviations ( SDs ) from three independent preparations . FPKMs of each gene in WT are shown in bottom graph .

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C qRT-PCR analysis of the highly transcribed PMA1, ADH1 and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph.


E ChIP using IgG - Sepharose or anti - Flag - agarose beads in strains expressing TAP - tagged Paf1 , Ubc9 , or Ulp1 or Flag - tagged Ulp2 . An untagged ( MHY500 ) was used as a negative control for immunoprecipitation of Ulp2 - Flag qPCR signals of indicated genes were quantitated normalized to an internal background control input . primer pairs used are indicated in ( D ) . presented as fold over background ; a value of 1 indicates no signal detected above background signal at a nontranscribed locus , as marked with horizontal . Error bars indicate SDs calculated from three independent preparations . Data information : Asterisks indicate statistically significant differences of Paf1 - TAP Ubc9 - TAP with Ulp1 - TAP Ulp2 - Flag with No tag in ( E ) using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations. Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with No tag in (E) using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


F Occupancy of Rpb3 Ulp2 at CUP1 gene was determined by ChIP in a expressing Flag - tagged Ulp2 using anti - Rpb3 antibody anti - Flag agarose beads Mock ' indicates use of G beads without added antibody . For CUP1 induction , were harvested at indicated time points after adding CuSO4 . " Pro " " ORF " represent positions of PCR fragments 1 2 from CUP1 gene Black bar indicates a value of 1 , background signal . Error bars , SD from four independent experiments . Data information : Asterisks indicate significant differences between uninduced induced in ( F ) using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 )

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F Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads Mock' indicates use of Protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. "Pro" and "ORF" represent the positions of PCR fragments 1 and 2 from the CUP1 gene Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments. Data information: Asterisks indicate significant differences between uninduced and induced cells in (F) using a two-tailed Student's t test (*, P <0.05; **, P <0.01)


G A graph of association of Ulp2 - Flag with genes classified by FPKM RPs ND indicate ribosomal genes genes not detected genes in our earlier - seq experiments , respectively . numbers above graph indicate total number of yeast genes in each expression category .

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G A percentage graph of association of Ulp2-Flag with genes classified by FPKM RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA-seq experiments, respectively. The numbers above the graph indicate the total number of yeast genes in each expression category.


H Average plot of Ulp2 - Flag occupancy at transcribed genes . values of y - axis indicate rescaled fold - enrichment , setting maximum occupancy to 1 minimum occupancy to 0 , x - axis indicates normalized from transcription start sites ( TSS ) transcription end sites ( TES ) . dotted lines indicate TSS TES . ChIP - seq data were obtained from duplicate experiments .

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H Average plot of Ulp2-Flag occupancy at transcribed genes. The values of the y-axis indicate rescaled fold-enrichment, setting the maximum occupancy to 1 and the minimum occupancy to 0, and the x-axis indicates normalized distance from transcription start sites (TSS) and transcription end sites (TES). The dotted lines indicate the TSS and TES. The ChIP-seq data were obtained from duplicate experiments.


A Sensitivity of indicated mutants to 6 - AU . strains carried a URA3 plasmid , pRS316 , were spotted on SD - Ura with or without 6 - AU ( 100 μg / ml ) ; plates were incubated for 2 - 4 days at 30 ° C . ULP2 ulp2 ( C624A ) represent ulp2Δ : : HIS3 containing either pRS314 - ULP2 - FLAG or pRS314 - ulp2 ( C624A ) - FLAG .

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A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.


ChIP analyses using anti - Rpb3 ( B ) antibodies in ulp2Δ Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP analyses using anti-Rpb3 (B) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


ChIP analyses using anti - SUMO ( C ) antibodies in ulp2Δ Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

ChIP analyses using anti-SUMO (C) antibodies in ulp2Δ cells Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


D qRT - PCR of CUP1 mRNA in ulp2Δ . Data were normalized to ACT1 mRNA . CUP1 gene induction was performed Error bars represent SD from three samples . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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D qRT-PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed Error bars represent the SD from three RNA samples. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


ChIP assays using anti - Rpb3 antibodies in ulp2Δ Pro " denotes # 1 PCR product of PMA1 , ADH1 PYK1 genes , while " ORF " indicates # 3 products of PMA1 # 2 of ADH1 PYK1 ; these are used in ensuing figures except where specified . Error bars indicate SD from four ( E ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 .

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ChIP assays using anti-Rpb3 antibodies in ulp2Δ cells Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes , while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01


ChIP assays using anti - SUMO ( F ) antibodies in ulp2Δ ; Pro " denotes # 1 PCR product of PMA1 , ADH1 PYK1 genes while " ORF " indicates # 3 products of PMA1 # 2 of ADH1 PYK1 ; these are used in ensuing figures except where specified . Error bars indicate SD from three ( F ) independent assays . Data information : Asterisks indicate statistically significant differences determined by pairwise comparisons between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP assays using anti-SUMO (F) antibodies in ulp2Δ cells ;Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from three (F) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


A Sensitivity to 6 - AU of indicated expressing chromosomally integrated ULP2 - Myc derivatives . Assays were performed schematic diagram below shows full - Ulp2 with a 9Myc epitope tag segments defining N - C - terminal domains catalytic ULP domain ( UD ) .

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A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).


B Immunoblot of Ulp2 - Myc derivatives expressed in

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B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strains


C ChIP of Flag - Ulp2 derivatives performed with same PCR probes W303a carrying pRS424 - GAL1 plasmids that expressed N - terminally Flag - tagged Ulp2 derivatives - specifically , full - , NTD ( 1 - 403 ) CTD ( 667 - 1034 ) - were grown to mid - log phase in SD - Trp medium containing 2 % galactose . Error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT , full of Ulp2 ( * , P < 0 . 05 ) .

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C ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05).


D Co - IP for interaction between Ulp2 - Myc derivatives Flag - H2B in strains transformed with a pRS314 plasmid expressing Flag - tagged H2B . Immunoprecipitated ( IP ) from anti - Myc agarose beads were analyzed by immunoblotting ( IB ) using anti - Myc or anti - Flag antibodies . upper middle panels show anti - Myc blotting anti - Flag blotting , respectively . of bound H2B were measured relative to INPUT H2B shown in lower panel .

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D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.


E association assays using strains After fractionation into ( pellet ) soluble ( supernatant ) components , fractions were analyzed by immunoblotting using indicated antibodies .

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E Chromatin association assays using strains After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies.


F Quantitation of association of Ulp2 - Myc derivatives in ( E ) . of Ulp2 in each fraction were measured by ImageJ then each signal was divided by total values for each Ulp2 derivative .

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F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.


A Immunoblot of immunoprecipitated Flag - tagged histone H2B with anti - Flag ( bottom panel ) or anti - SUMO ( upper panel ) antibodies in WT , ulp2Δ , SUMO - KR or ulp2Δ SUMO - KR strains carrying empty vector or pRS426 - FLAG - HTB1 . One , two or three asterisks represent mono , di polysumoylated histones , respectively . Note that apparent diSUMO - H2B species ( * * ) in ulp2Δ SUMO - KR migrates more slowly than H2B linked to a SUMO dimer , suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines ( lane 8 ; also see weaker band in lane 6 ) .

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A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).


B Yeast strains of indicated genotypes carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; were grown at 30 ° C for 3 days .

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B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.


C Mutant htb1 - K123R ulp2Δ htb1 - K123R ULP2 strains with a pRS316 - ULP2 cover plasmid were grown on indicated plates at 30 ° C for 2 - 3 days . Three double mutant isolates were tested in parallel .

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C Mutant htb1-K123R ulp2Δ and htb1-K123R ULP2 strains with a pRS316-ULP2 cover plasmid were grown on the indicated plates at 30°C for 2-3 days. Three double mutant isolates were tested in parallel.


D Mutant ulp2Δ bre1Δ strains carrying a pRS426 - ULP2 ( URA3 ) cover plasmid indicated plasmids were grown on SD - Ura SD + 5 - FOA at 30 ° C for 2 - 3 days .

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D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.


E Yeast strains of indicated genotypes carrying a YCplac33 - ULP2 cover plasmid were streaked on SD + 5 - FOA to evict YCplac33 - ULP2 ; were grown at 30 ° C for 3 days .

E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.


A Immunoblot of immunoprecipitated Flag - tagged histone H2B using anti - Flag or anti - SUMO antibodies in indicated mutants , upper lower panels show Flag - H2B - SUMO Flag - H2B - ub , respectively . One , two or three asterisks indicate mono - , di - , poly - sumoylated histones ( upper panel ) , respectively , an arrowhead bullet point represent mono - ubiquitylated H2B , a non - specific band , respectively .

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A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.


B Immunoblot of sumoylated in extracts prepared from strains Anti - Flag blotting for Flag - H2B was used to verify similar loading .

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B Immunoblot assay of sumoylated proteins in extracts prepared from the strains Anti-Flag blotting for Flag-H2B was used to verify the similar loading.


C double immunoprecipitation ( ChDIP ) of Flag - H2B - ub in indicated strains expressing Flag - H2B . first ChIP was performed with anti - Flag agarose , eluted samples were immunoprecipitated with an antibody against ubiquitin . PCR signals from indicated genes were normalized to an internal control input . error bars indicate SD from three independent samples . Data information : Asterisks indicate statistically significant differences compared with WT in * , P < 0 . 05 ; * * , P < 0 .

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C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples. Data information: Asterisks indicate statistically significant differences compared with WT in *, P <0.05; **, P <0.01


D ChDIP of Flag - H2B - SUMO in indicated strains expressing both Flag - H2B HA - SUMO . samples were sequentially immunoprecipitated with anti - HA agarose followed by anti - Flag agarose . PCR signals were normalized to input . error bars indicate SD from three independent experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 .

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D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01


E ChDIP of Flag - H2B - SUMO in indicated mutant strains during CUP1 gene induction CUP1 induction was performed CUP1 OFF ON indicate uninduced induced ( 5 min ) conditions , respectively . error bars represent SD from three independent ChDIP assays . Data information : Asterisks indicate significant differences between uninduced induced in ( E ) ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction The CUP1 induction was performed CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays. Data information: Asterisks indicate significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01).


F Immunoblot of Flag - tagged Ulp2 in indicated strains . Anti - PGK blotting shows similar loading .

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F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading.


ChIP assays in indicated strains expressing Flag - tagged Ulp2 error bars indicate SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT * , P < 0 . 05 ; * * , P < 0 .

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ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT *, P <0.05; **, P <0.01


ChIP assays in indicated strains expressing Flag - tagged Ulp2 error bars indicate SD from three experiments . Data information : Asterisks indicate statistically significant differences compared with WT ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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ChIP assays in the indicated strains expressing Flag-tagged Ulp2 The error bars indicate the SD from three experiments. Data information: Asterisks indicate statistically significant differences compared with WT (*, P <0.05; **, P <0.01).


A of CTD S2 - P relative to Rpb3 at 3 ' ORF regions was analyzed by ChIP using anti - S2 - P anti - Rpb3 antibodies . error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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A The ratio of CTD S2-P relative to Rpb3 at 3' ORF regions was analyzed by ChIP using anti-S2-P and anti-Rpb3 antibodies. The error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


B A ulp2Δ single mutant three isolates of ulp2Δ ctk1Δ with YCplac33 - ULP2 were grown on SD - Ura or SD + 5 - FOA plates at 30 ° C for 2 - 3 days .

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B A ulp2Δ single mutant and three isolates of ulp2Δ ctk1Δ with YCplac33-ULP2 were grown on SD-Ura or SD+5-FOA plates at 30°C for 2-3 days.


E ChIP analyses of Ctk1 - TAP in indicated strains . ORF of TFIID - dominated genes indicates position 2 of PCR fragments from UTH1 ATP2 genes Error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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E ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


F Co - IP of Ctk1 histone H2B interaction . WT ulp2Δ strains expressing indicated tagged were precipitated with IgG - Sepharose . IP INPUT were subjected to immunoblotting with PAP ( Ctk1 - TAP ) anti - Flag ( Flag - H2B ) antibodies . signals were quantified relative to PGK then normalized to WT value .

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F Co-IP analysis of Ctk1 and histone H2B interaction. WT and ulp2Δ strains expressing the indicated tagged proteins were precipitated with IgG-Sepharose. IP and INPUT proteins were subjected to immunoblotting with PAP (Ctk1-TAP) and anti-Flag (Flag-H2B) antibodies. Protein signals were quantified relative to PGK and then normalized to the WT value.


G ChDIP of Flag - H2B - SUMO in indicated strains " Pro " " ORF " of UTH1 ATP2 genes indicate positions 1 2 of PCR fragments , respectively error bars indicate SD from three independent ChDIPs . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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G ChDIP of Flag-H2B-SUMO in the indicated strains The "Pro" and "ORF" of UTH1 and ATP2 genes indicate positions 1 and 2 of PCR fragments, respectively The error bars indicate the SD from three independent ChDIPs. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


H Sensitivity to 6 - AU of indicated htb1 - 1 htb2 - 1 ( null alleles for both chromosomal H2B genes ) strains carrying pRS314 plasmids that express Flag - H2B or 2SUMO - H2B .

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H Sensitivity to 6-AU of indicated htb1-1 htb2-1 (null alleles for both chromosomal H2B genes) strains carrying pRS314 plasmids that express Flag-H2B or 2SUMO-H2B.


I , J ChIP analyses of indicated strains Occupancy of Rpb3 ( I ) of CTD S2 - P relative to Rpb3 ( J ) at 3 ' ORF regions of indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

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I, J ChIP analyses of the indicated strains Occupancy of Rpb3 (I) and the ratio of CTD S2-P relative to Rpb3 (J) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


K ChIP analyses of Ctk1 - TAP in indicated strains . ORF of TFIID - dominated genes indicates position 2 of PCR fragments from UTH1 ATP2 genes Error bars represent SD from triplicate experiments . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

K ChIP analyses of Ctk1-TAP in the indicated strains. The ORF of TFIID-dominated genes indicates position 2 of PCR fragments from the UTH1 and ATP2 genes Error bars represent the SD from triplicate experiments. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


L Immunoblot of TAP - tagged Ctk1 in extracts prepared from strains Anti - PGK blotting shows similar loading .

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L Immunoblot analysis of TAP-tagged Ctk1 proteins in extracts prepared from the strains Anti-PGK blotting shows similar loading.


M , N ChIP analyses of indicated strains Occupancy of Rpb3 ( M ) of CTD S2 - P relative to Rpb3 ( N ) at 3 ' ORF regions of indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses . Data information : Asterisks represent statistically significant differences between WT ulp2Δ using a two - Student ' s t ( * , P < 0 . 05 ; * * , P < 0 . 01 ) .

M, N ChIP analyses of the indicated strains Occupancy of Rpb3 (M) and the ratio of CTD S2-P relative to Rpb3 (N) at 3' ORF regions of the indicated genes were individually analyzed by ChIP Error bars show SD from three independent analyses. Data information: Asterisks represent statistically significant differences between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01).


( D ) showing expression change of representative NF - κB target genes from genes with significantly changed expression ( fold change > 2 ) . data represent log2 ( gene FPKM ) normalized for each row . - seq data is from 2 independent experiments .

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(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 of human TNF - α IL - 2 in sh - NC sh - p38IP Jurkat E6 . 1 stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for 6 hours ( average frequency of positive YFP + in sh - NC without stimulation = 1 ) .

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(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 of IL - 2 IL - 6 production in culture supernatants of human PBMCs transfected with si - NC or si - p38IP , then stimulated with 10 μg / ml anti - CD3 plus 10 μg / ml anti - CD28 ( αCD3 / CD28 ) for 24 hours .

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(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 H ) Real - time PCR of TNF - α , IL - 6 IL - 1β ( G ) of IFN - β , ISG54 ISG56 ( H ) mRNA production in RAW264 . 7 transfected with si - NC si - mp38IP - 1 then stimulated with 500 ng / ml LPS for indicated times ( average mRNA production in si - NC at 0 hour = 1 ) .

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(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 of TNF - α , IL - 6 , IL - 1β IFN - β production in culture supernatants of human PBMCs transfected with si - NC si - p38IP then stimulated with 1μg / mL LPS for 24 hours .

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(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.


( A ) Immunoblot with indicated antibodies of total lysates of sh - NC sh - p38IP Jurkat E6 . 1 stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times . Actin served as a loading control ( throughout ) .

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(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 of p65 translocation of sh - NC sh - p38IP Jurkat E6 . 1 T stimulated with 50 ng / ml PMA plus 1µM ionomycin for indicated times . p65 translocation was indicated by intensities between p65 whole p65 from each . * * * P < 0 . 001 ( two - unpaired t - , mean s . e . m . ) .

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(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 of sh - NC sh - p38IP Jurkat E6 . 1 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 - unpaired t - , mean s . e . m . ) .

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(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 with indicated antibodies of total lysates of RAW264 . 7 transfected with si - NC si - mp38IP - 1 then stimulated with 500 ng / ml LPS for indicated times .

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(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.


( A ) Fragmentation spectrum of p38IP peptide identified in TAK1 immunoprecipitated from Jurkat TAg by liquid chromatography - tandem mass spectrometry . m / z , mass / charge .

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(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 were transfected with YFP - p38IP , lysates aliquots were immunoprecipitated with IgG , anti - TAB1 , anti - TAB2 anti - TAK1 respectively , then immunoblotted with indicated antibodies .

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(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 were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times lysed , followed byTAK1 immunoprecipitation . TAK1 beads were washed three times with lysis buffer two times with kinase buffer , then resuspended in kinase buffer containing 100 μM ATP 1μg recombinant GST - IKKβ ( 111 - 250aa ) as substrate to perform in vitro kinase , then immunoblot with p - IKKβ antibody .

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(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 were transfected with cMyc vector ( - ) or increasing amounts of cMyc - p38IP ( wedge ) , 24 hours post transfection , lysates were immunoprecipitated with IgG or anti - TAB2 then immunoblotted with anti - TAK1 anti - TAB2 . Expression of cMyc - p38IP was determined in immunoblotted lysates with anti - cMyc .

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(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 were transfected with HA vector ( - ) or increasing amount of HA - p38IP ( wedge ) , 24 hours post transfection , lysates were immunoprecipitated with IgG or anti - TAK1 then immunoblotted with anti - TAB2 , anti - TAB1 , anti - HA anti - TAK1 . Expression of HA - p38IP was determined in immunoblotted lysates with anti - HA .

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(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 sh - p38IP Jurkat E6 . 1 T were stimulated with 10 μg / ml anti - CD3 plus 2 μg / ml anti - CD28 for indicated times , lysates were immunoprecipitated with anti - TAB2 immunoblotted with anti - TAK1 anti - TAB2 .

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(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 transfected with cMyc vector or cMyc - p38IP were stimulated with 10μg / ml anti - CD3 for indicated times , then were lysed immunoprecipitated with anti - TAK1 antibody immunoblotted with anti - K63Ub , anti - cMyc anti - TAK1 antibodies . Lysates were blotted with indicated antibodies , actin served as loading control .

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(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 sh - p38IP Jurkat E6 . 1 T stimulated as in ( F ) were lysed immunoprecipitated with anti - TAK1 immunoblotted with anti - K63Ub anti - TAK1 . Lysates were immunoblotted with indicated antibodies .

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(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 transfected stimulated as in ( H ) were lysed immunoprecipitated with anti - TAB2 immunoblotted with anti - K63Ub anti - TAB2 .

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(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 sh - p38IP Jurkat E6 . 1 stimulated as in ( F ) were lysed immunoprecipitated with anti - TAB2 immunoblotted with anti - K63Ub anti - TAB2 . Data ( B - F , H - K ) are representatives of at least three independent experiments .

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(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.


( A ) Jurkat E6 . 1 T were stimulated with 10 μg / ml anti - CD3 2 μg / ml anti - CD28 for indicated times , lysates were immunoprecipitated immunoblotted as indicated . relative of IP bands was quantified by densitometry is shown below graph .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

(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 transfected with Flag - tagged TAB2 ( open arrow ) or TAB2 - ub ( solid arrow ) were lysed immunoprecipitated with anti - Flag , then immunoblotted with indicated antibodies . asterisk indicates a non - specific band . relative of IP bands was quantified by densitometry is presented in right .

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(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 co - transfected with HA - tagged p38IP together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) were lysed immunoprecipitated with anti - cMyc , then immunoblotted with indicated antibodies . Asterisk indicates non - specific bands . relative of IP bands was quantified by densitometry is presented in right .

O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

(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 co - transfected with HA - tagged p38IP , Flag - tagged TAB2 - ub , together with cMyc - tagged TAK1 ( open arrow ) or ub - TAK1 ( solid arrow ) , were lysed immunoprecipitated with anti - cMyc , then immunoblotted with indicated antibodies . relative of IP bands was quantified by densitometry is presented in right .

O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O

(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.


( A ) GSH - Sepharose beads - immobilized full or truncated GST - TAK1 were incubated with K63 - linked polyUb chains in 300 μl binding buffer , followed by washing resolving in SDS - PAGE , then immunoblotted with anti - ub . GST - fused NEMO serve as positive control NEMO L329P mutant as negative control . blotted was stained with Ponceau S to demonstrate amount of input GST - fused ( lower panel ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O

(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 D ) Immobilized GST - TAB2 was pre - incubated with K63 - linked polyUb chains then eluted from beads , followed by incubation with eluted GST - TAK1 GST - p38IP as shown in ( B ) . Incubation mixture were immunoprecipitated with anti - TAB2 or anti - TAK1 , then immunoblotted with anti - ub , anti - TAB2 , anti - TAK1 anti - p38IP . ( E ) Statistical of polyUb binding to TAB2 or TAK1 in ( C D ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( two - unpaired t - , mean s . e . m . ) .

O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 , then immunoblotted with anti - TAB2 anti - TAK1 . relative of immunoprecipitated TAK1 was calculated shown in right . * * * P < 0 . 001 ; n . s . , not significant ( two - unpaired t - , mean s . e . m . ) .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.).