Extended Data Fig. 2: Impact of MG132 treatment and Kless mutation on CTLA-4 ubiquitylation.
a). Transendocytosis was carried out using CD80-GFP or CD86-GFP expressing CHO cells or CHO cells with no ligand (NL) for the times shown and whole cell lysates were blotted for GFP (ligand), CTLA4 (C-term), with membranes stripped and reprobed for tubulin. CTLA-4 increases in Mw are highlighted (red box). b). CTLA-4 lacking cytoplasmic lysine residues (CTLA-4 Kless) was used in transendocytosis assays with CHO cells expressing CD80, CD86 or CHO cells with no ligand (NL) for times indicated and whole cell lysates blotted for GFP (ligand), and CTLA-4. Lysates were blotted for tubulin as a sample processing control. c). The impact of Kless mutation on ubiquitylation of CTLA-4 expressed in Jurkat T cells. Transendocytosis of DG75 B cells expressing CD80-GFP or CD86-GFP was carried out for 6 hours, followed by lysis and immunoprecipitation of total ubiquitin (ubiquitin trap). Blots were then probed for CTLA-4 expression using anti-CTLA4 antibody (C-term) and GFP (ligand). Whole cell lysates (WCL) were also blotted using anti-CTLA4, and tubulin to control for protein loading. All data is representative of at least 3 independent experiments.
