Figure 5.

Fas clustering and capping in WR/Fas-SM(−) and WR/Fas-SMS1 cells. Time kinetics (A) and dose dependency (B) of Fas multimer formation. 4 × 10⁷ cells were stimulated with 50 ng/ml CH11 for the indicated time (A), or stimulated for 10 min with the indicated concentration of CH11 (B). After stimulation, cell pellets were snap frozen and treated at 45°C for 1 h in 300 μl of a 1% NP-40–treating solution. DNA in the samples was sheared with a 25-gauge needle, and solubilized samples were loaded on 12% SDS-PAGE. Fas multimers were detected with antibody to the intracellular death domain of human Fas (3D5). Data are representative of more than three independent experiments. (C) Activation-induced capping of Fas. Cells were stained for 20 min with 50 ng/ml CH11 at 4°C. Afterwards, capping was induced by warming cells to 37°C for 30 min in a water bath with mild agitation. Cells were harvested at the indicated times and fixed with 4% paraformaldehyde for 20 min at 22°C. Fixed cells were washed twice and mounted in FITC-conjugated secondary antibody. Fluorescence was detected using a confocal microscope equipped with a SPOT digital camera. The data are representative of more than five experiments. Arrowheads indicate Fas capping. (D) Time kinetics of activation-induced Fas capping. Capping was induced for the indicated time, and cells with Fas clustering were counted by two independent observers. Percentages of capping were calculated in 150–200 total cells. These results are the mean of three independent experiments. Error bars represent SEM. *, P < 0.01.