Ubiquitylation of CTLA4 on Lys203 and Lys213 is responsible for its rapid turnover. (A) Depiction of the cytoplasmic tail of CTLA4 and the lysine mutants analyzed in this study. (B) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA WT or lysine mutant cells were transfected for 72 h with non-targeting (NT1) or USP8 siRNA. 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. (C) Quantification of ubiquitylated lysine mutant CTLA4-HA relative to WT for data represented in B. Ubiquitylated CTLA4-HA is shown normalized to immunoprecipitated CTLA4-HA. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (D) Representative western blots of HeLa S3 Flp-In CTLA4-HA WT, K-null, and K203R,K213R double lysine mutant cells treated with CHX for indicated times before lysis. (E) Quantification of CTLA4-HA turnover for data represented in D. The half-life of CTLA4-HA was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. Source data are available for this figure: SourceData F9.