FIG. 8.
Formation of SDS-stable CD95 microaggregates is independent of actin and caspase activity. (A) CD95 was immunoprecipitated from either 107 unstimulated (U) or anti-CD95 stimulated (5 min) SKW6.4 cells that were left untreated (S) or that had been pretreated with 50 μM zVAD-fmk (Z). Immunoprecipitates were subjected to SDS-PAGE (12% polyacrylamide) and immunoblotted with anti-caspase 8 and anti-FADD antibodies. p43 and p41 are cleavage intermediates of activated caspase 8. DISC formation was also unaffected by zVAD-fmk when cells were stimulated with anti-CD95 for 60 min (data not shown). (B) SKW6.4 cells were incubated with anti-CD95 and left unstimulated (time 0) or stimulated for 60 min (60′) at 37°C as described in Fig. 2C after pretreatment with 50 μM zVAD-fmk. 0, mock treatment with 0.5% dimethyl sulfoxide (DMSO) alone. Samples were analyzed by fluorescence microscopy. (C) Ligand-induced internalization of SKW6.4 cells or K50 cells untreated or pretreated with zIETD-fmk for 60 min was quantified as described in Fig. 3C. (D) A total of 106 SKW6.4 and H9 cells stimulated with anti-CD95 (20 min) were left untreated (U) or had been pretreated with 5 μM Ltn A (L) or 50 μM zVAD-fmk (Z). Unstimulated cells were analyzed as a control (C). Cell lysates were subjected to SDS-PAGE (10% polyacrylamide) and immunoblotted with anti-CD95 (C20) antibody. CD95hi corresponds to SDS-stable CD95 microaggregates migrating at around 220 kDa during SDS-PAGE.