Figure 1.

Cell surface half-life (A) and endocytosis (B) of IL2Rβ on stably transfected ts20 mutant cells. Adherent ts20β cells were harvested using a 10-min incubation with PBS containing 5 mM EDTA at 33°C and, after one wash, preincubated for 1 h at permissive (30°C) or nonpermissive (42°C) temperature before and during the assays. (A) Cell surface expression of IL2Rβ on cells treated for different times with of 50 μM of cycloheximide was assayed by flow cytometry by using mAb 341 as described in MATERIALS AND METHODS. (B) For internalization assays, after 1-h preincubation at 30°C or 42°C, cells were labeled with the antibody at 4°C for 1 h, washed once at 4°C, and incubated for the indicated times at permissive or nonpermissive temperature. Disappearance of anti-IL2Rβ mAb from the cell surface upon internalization was assessed by cytofluorimetry and the percentage of internalized antibody was calculated. The y-axis is the ratio of the antibody internalized to the amount of antibody bound to the cell surface at each time point. The slope of this plot is the internalization rate constant (Wiley and Cunningham, 1982). The results presented in A and B are the means ± SE of at least three independent experiments.