Panel A shows a line graph tracking relative CTLA-4 degradation across multiple experimental time points. Panel B shows bar graphs comparing relative CTLA-4 expression after inhibitor treatments under different conditions. Panel C shows histograms illustrating CTLA-4 expression changes following chloroquine treatment in peripheral blood mononuclear cells. Panel D shows bar graphs quantifying CTLA-4 expression levels after chloroquine treatment relative controls. Panel E shows a bar graph depicting CTLA-4 fold-change following chloroquine treatment across conditions. Panel F shows bar graphs comparing CTLA-4 fold-change after inhibitor treatments and experimental manipulations. Panel G shows immunoblot images assessing endoplasmic reticulum stress marker expression in transfected cells.
CTLA4 S172P/S172P variant accelerates the lysosomal degradation of CTLA-4. (A) PBMCs were treated with 30 μg/ml CHX for up to 4 h at 37°C and stained for total CTLA-4. The graph shows total CTLA-4 expression relative to 0 h, indicating relative CTLA-4 degradation. (B) Relative CTLA-4 expression after 6 h of CHX treatment with or without chloroquine and MG-132, normalized to HCs. (C) Representative histograms showing CTLA-4 expression in PBMCs treated with chloroquine for 6 h compared with untreated conditions. (D) Bar graphs illustrating CTLA-4 expression after 6 h of chloroquine treatment, normalized to HC. (E) Fold change in CTLA-4 expression after 6 h of chloroquine treatment, normalized to untreated conditions. (F) Bar graphs showing CTLA-4 fold change following 6 h of CHX treatment with or without chloroquine or MG-132, normalized to untreated conditions. (G) Immunoblot analysis of the ER stress marker BiP in cells transfected with WT CTLA-4 and two independent S172P mutant clones. GAPDH was used as a loading control. Statistical significance was assessed using two-way ANOVA with Tukey’s post hoc test for Panels (A, B, and D). In panel F, one-way ANOVA with Tukey’s post hoc test was used to analyze significance. ns = nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. ER, endoplasmic reticulum; HC, healthy control; WT, wild-type; Mut, mutant; col, colony; CHX, cycloheximide; CQ, chloroquine. Source data are available for this figure: SourceData F4.