Fig 2: Endocytosis analysis of CTLA-4 for the WT, G199R, and ΔC mutant. (A) Diagram of the antibody labeling strategy for flow cytometry experiments in (B). (B) Jurkat cells expressing different CTLA-4 were labeled with anti-CTLA-4 APC at 37 °C for 30 min, followed by labeling surface CTLA-4 on ice (4 °C) with a fluorescently conjugated anti-mouse secondary antibody (PE) for 1 h. Cells were then analyzed by flow cytometry.

Fig 3: Biochemical and functional comparison between the WT, G199R, and ΔC mutant. (A) Amino acid sequence of the cytoplasmic domain of CTLA-4 and G199R mutant. Red letters indicate the tyrosine-containing motifs (Y201VKM) that are conserved between species. Domains: TM, transmembrane; CTD, cytoplasmic domain. (B) Bar graph summarizing IL-2 release from a 24-h Jurkat-Raji coculture with different transduced CTLA-4 mutants into Jurkat cells. The data shown are representative of the results from at least three independent experiments. (C) Cellular level of CTLA-4 or mutants in Jurkat cells determined by western blot. Samples were run under non-reducing conditions. (D) Surface level of the WT (blue), G199R (red), and ΔC mutant (yellow) in Jurkat cells determined by flow-cytometric analysis using APC-conjugated anti-CTLA-4 mAb. The control (grey) corresponds to cells without any antibody treatment.