FIGURE 1.

Endocytosis of CTLA-4. A, CTLA-4 C-terminal sequence alignments show the truncations used in this study. B, CHO cells expressing either wild type (wt), Δ13, or Δ23 CTLA-4 were incubated with unlabeled anti-CTLA-4 at 37 °C for 30 min. Cells were then cooled to 4 °C, and CTLA-4 that remained on the cell surface were stained with a fluorescently labeled secondary antibody (red). Cells were then fixed, permeabilized, and stained with a different secondary antibody (green) and imaged by confocal microscopy. The ratio of plasma membrane to internalized CTLA-4 fluorescence (PM/I) was calculated by outlining cells in ImageJ and is shown in the right panel. C, diagram of the antibody labeling strategy for flow cytometry experiments in D and E. D, CHO cells expressing different CTLA-4 truncations were labeled with anti-CTLA-4 PE at 37 °C for 30 min followed by labeling surface CTLA-4 on ice (4 °C) with a fluorescently conjugated anti-mouse secondary antibody. Cells were analyzed by flow cytometry. E, CHO cells expressing CD28, CTLA-4 wt, or CTLA-4 AVKM were labeled as described in C and D and analyzed by flow cytometry. Red lines indicate the staining ratio of Δ23 (D) or CD28 (E) as a guide for the expected ratio for a cell surface protein. F, CHO cells expressing wild-type CTLA-4 were labeled at 4 °C with anti-CTLA-4 PE to label surface CTLA-4. Cells were then warmed to 37 °C for the time indicated, and CTLA-4 remaining on the surface was detected on ice with a fluorescently conjugated anti-mouse secondary antibody. The time course of surface labeling is plotted against initial labeling.