Lysosomal degradation of CTLA4 requires a functional ubiquitin conjugation cascade. (A) Representative western blots of A2058, NCI-H520, and HeLa S3 Flp-In CTLA4-HA cells treated with Cycloheximide (CHX, 100 µg/ml) for indicated times prior to lysis. (B) Quantification of data represented in A. The half-life of CTLA4 in different cell lines was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. (C and E) Representative western blots of A2058 and NCI-H520 cells treated with Concanamycin (ConcA, 100 nM), Epoxomicin (Epo, 100 nM), or TAK-243 (TAK, 1 µM) for 15 min before the addition of CHX for 1 h (C, A2058) or 2 h (E, NCI-H520). (D and F) Quantification of CTLA4 levels after treatment with indicated inhibitors relative to control for data represented in C and E. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. One-way ANOVA and Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01, ****P < 0.0001. Source data are available for this figure: SourceData F1.