AP2 depletion delays CTLA4 turnover and reduces CTLA4 ubiquitylation in the absence of USP8. (A and D) Representative western blots following treatment with Cycloheximide (CHX; 100 µg/ml) for indicated times in HeLa S3 Flp-In CTLA4-HA (A) and A2058 (D) transfected with indicated siRNAs. Cells were transfected with non-targeting (NT1) or AP2 siRNA targeting the µ2 subunit and reseeded for a second round of transfection 24 h later with NT1, AP2, and/or USP8 siRNA. The cells were left for a further 72 h before lysis. The last lane represents untransfected control. *non-specific signal. (B and E) Quantification of basal CTLA4 levels normalized to untransfected control for data represented in A and D. Error bars show SD from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. (C and F) Quantification of CTLA4 levels following treatment with Cycloheximide for data represented in A and D. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. (G and H) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected as in A and D. IB: immunoblot. *non-specific signal. (I and J) Quantification of ubiquitylated CTLA4 relative to total CTLA4 levels for data represented in G and H. Individual data points from two (I) or three (J) independent, color-coded experiments are shown. Error bars indicate range (n = 2) or SD (n = 3). Source data are available for this figure: SourceData F5.