Mutation of K203 and K213 delays CTLA4 turnover. (A) Representative western blots showing increased stability of CTLA4-HA K-null and K203R, K213R mutants. HeLa S3 Flp-In CTLA4-HA WT and indicated lysine mutants were treated with Cycloheximide (CHX) for 2 h before lysis. (B) Quantification of CTLA4-HA remaining following CHX treatment. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. (C) Representative western blots of HeLa S3 Flp-In CTLA4-HA WT, K-null and K203R, K213R double lysine mutants treated for 4 h with Cycloheximide (CHX) alone or together with Concanamycin A (ConcA) or Epoxomicin (Epo) prior to lysis. (D) Quantification of CTLA4-HA remaining normalised to control treated cells for data represented in C. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. Two-way ANOVA multiple comparisons with uncorrected Fisher’s LSD, *P < 0.05, **P < 0.01. (E and F) Representative confocal images of CTLA4-HA WT, K-null, and K203R,K213R mutants co-stained with EEA1 (E) or LAMP1 (F). Scale bar = 15 µm (main figure) and 5 µm (inset). (G and H) Co-localization analysis of CTLA4-HA WT, K-null and K203R,K213R, and with EEA1 (G) and LAMP1 (H). Graphs show Pearson’s coefficients or Mander’s coefficients. Error bars indicate SD for three independent, color-coded experiments. Opaque circles with dark outlines correspond to the mean value from each experiment. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. Source data are available for this figure: SourceData FS4.