Fig. 2: CD80 engagement drives CTLA-4 ubiquitylation.
a, Immunoblot analysis of TE experiments using CD80–GFP- and CD86–GFP-expressing CHO cells compared with cells with no ligand (NL). After TE for the times shown, whole-cell lysates (WCL) from combined CTLA-4 and ligand-expressing cells were directly blotted using anti-CTLA-4 (C-term) and anti-GFP (ligand) antibodies. Lysates were also blotted for tubulin as a sample-processing control. b, Quantification of anti-CTLA-4 (C-term) blots showing >25-kDa smear density relative to the 25-kDa band after CD80 or CD86 TE. The graph compares CTLA-4 WT or CTLA-4 lacking cytoplasmic lysine residues (K-less). Analysis was performed in Image Lab (BioRad) from four independent experiments. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparison correction. All data are presented as mean ± s.d. and show individual data points. ****P < 0.0001. c, CHO TE experiments performed for 4 h and IP carried out via CD80–GFP or CD86–GFP. Precipitates from WT CTLA-4, K-less and CTLA-4 lacking a cytoplasmic domain (Δ36) were blotted for ubiquitin, CTLA-4 (C-term) and ligand (GFP). d, TE using CHO–CD80/86-CTLA-4 (WT/K-less) was carried out for the times indicated, subjected to ubiquitin IP, followed by blotting for CTLA-4 (C-term) and CD80/86 (GFP). e, CD80 and CD86 TE experiments performed with CTLA-4-expressing Jurkat T cells and DG-75 B cells expressing CD80–GFP or CD86–GFP at a 1:1 ratio. At the times indicated, ubiquitin was precipitated and then blotted for CTLA-4 and ligand–GFP. f, CD80 and CD86 TE experiments carried out using DG-75–GFP ligand-expressing cells and unmodified primary human Treg cells for 5 h followed by ubiquitin precipitation and blotting for CTLA-4 and ligand. All data represent a minimum of three similar experiments. The increased Mr of CTLA-4 is highlighted by a red box. For IPs, WCLs from all experiments were blotted for tubulin to control for protein loading.
