Catalytic activity and endosomal localization of USP8 are essential for CTLA4 degradation and ubiquitylation. (A) Representative confocal images of HeLa S3 Flp-In CTLA4-HA transfected for 72 h with NT1 or USP8 siRNAs before Cycloheximide (CHX) treatment for indicated times. Cells were fixed and stained for HA. Scale bar = 15 µm. (B) GFP-tagged USP8 siRNA-resistant (USP8*) constructs used in this study. MIT, Microtubule interacting; SBD: SH3 domain binding motif; RHOD, Rhodanese homology domain; USP, Ubiquitin specific protease - catalytic domain. (C) Representative confocal images of HeLa S3 Flp-In CTLA4-HA cells transfected with non-targeting (NT1) or USP8 siRNA and GFP, siRNA-resistant GFP-tagged USP8 (USP8*), catalytically inactive USP8 (C786S*) or ∆MIT-USP8. Cells were treated for 2 h with CHX prior to fixation and staining for HA. Scale bar = 15 µm. (D) Quantification of cells showing rescued phenotypes calculated for data represented in C. Individual data points from 3 (∆MIT), 6 (C786S), or 7 (GFP and USP8*) independent, color-coded experiments are shown. Error bars indicate the SD. Total number of cells analyzed: GFP (599); USP8* (666); C786S (486); ∆MIT (322). One-way ANOVA and Dunnett’s multiple comparison test, ***P < 0.001 and ****P < 0.0001. (E) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected with NT1 or USP8 siRNA and either GFP or GFP-tagged siRNA-resistant USP8 constructs as in C. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. IB: Immunoblot. (F) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in E. Ubiquitylated CTLA4-HA was normalized to total CTLA4-HA pulled down. Individual data points from two (∆MIT) or five independent, color-coded experiments are shown. Error bars indicate the SD (n = 5) or range (n = 2). One-way ANOVA and Dunnett’s multiple comparison test,****P < 0.0001. Source data are available for this figure: SourceData F8.
Figure 8.

Catalytic activity and endosomal localization of USP8 are essential for CTLA4 degradation and ubiquitylation. (A) Representative confocal images of HeLa S3 Flp-In CTLA4-HA transfected for 72 h with NT1 or USP8 siRNAs before Cycloheximide (CHX) treatment for indicated times. Cells were fixed and stained for HA. Scale bar = 15 µm. (B) GFP-tagged USP8 siRNA-resistant (USP8*) constructs used in this study. MIT, Microtubule interacting; SBD: SH3 domain binding motif; RHOD, Rhodanese homology domain; USP, Ubiquitin specific protease - catalytic domain. (C) Representative confocal images of HeLa S3 Flp-In CTLA4-HA cells transfected with non-targeting (NT1) or USP8 siRNA and GFP, siRNA-resistant GFP-tagged USP8 (USP8*), catalytically inactive USP8 (C786S*) or ∆MIT-USP8. Cells were treated for 2 h with CHX prior to fixation and staining for HA. Scale bar = 15 µm. (D) Quantification of cells showing rescued phenotypes calculated for data represented in C. Individual data points from 3 (∆MIT), 6 (C786S), or 7 (GFP and USP8*) independent, color-coded experiments are shown. Error bars indicate the SD. Total number of cells analyzed: GFP (599); USP8* (666); C786S (486); ∆MIT (322). One-way ANOVA and Dunnett’s multiple comparison test, ***P < 0.001 and ****P < 0.0001. (E) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected with NT1 or USP8 siRNA and either GFP or GFP-tagged siRNA-resistant USP8 constructs as in C. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. IB: Immunoblot. (F) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in E. Ubiquitylated CTLA4-HA was normalized to total CTLA4-HA pulled down. Individual data points from two (∆MIT) or five independent, color-coded experiments are shown. Error bars indicate the SD (n = 5) or range (n = 2). One-way ANOVA and Dunnett’s multiple comparison test,****P < 0.0001. Source data are available for this figure: SourceData F8.

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