Regulation of CTLA4 ubiquitylation by USP8 is dependent on HD-PTP. (A) Representative western blots of USP8 co-immunoprecipitated with CTLA4-HA. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected for 72 h with non-targeting (NT1), USP8 or HD-PTP siRNA. Cells were lysed and lysates were subjected to HA-immunoprecipitation (IP; IB: Immunoblot). (B) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected as in A. 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. (C) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in B. Ubiquitylated CTLA4-HA was normalised to total CTLA4-HA pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (D) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected as in A and B and cell lysates subjected to TUBES pulldown. (E) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in D. Ubiquitylated CTLA4(-HA) was normalized to total ubiquitin pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (F) Representative western blots of CHX chase in HeLa S3 Flp-In CTLA4-HA and A2058 cells following transfection with non-targeting (NT1) or HD-PTP siRNA. Cells were treated with CHX for indicated times before lysis. (G) The half-life of CTLA4 was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. Source data are available for this figure: SourceData F7.