FIGURE 8.

PMA inhibits Fas trafficking to cell surface. A, JFSD9 cells (reconstituted with Fas-S peptide) were treated with 500 ng/ml CH-11 (lanes 2–5 and 7–10) for the indicated lengths of time in the absence (lanes 2–5) or presence (lanes 7–10) of 20 nm PMA. For the 0-min time point, cells were treated with diluent (lane 1) or PMA for 90 min (lane 6). After cell surface proteins were biotinylated and cell lysates were prepared, S peptide-tagged Fas was recovered with anti-S peptide antibody coupled to protein G-agarose. Immunoprecipitates were probed with peroxidase-coupled streptavidin (top, cell surface Fas), anti-S peptide antibody (middle, total cellular Fas), or anti-FADD antibody. B, after Jurkat cells were treated with 250 ng/ml CH-11 (circles) or control IgM (squares) for the indicated length of time in the absence (open symbols) or presence (closed symbols) of 20 nm PMA, cells were cooled, washed labeled with APC-conjugated anti-mouse IgM, and analyzed as described in the legend to Fig. 6C. MFI, mean fluorescence intensity.