Gadd45β has no effect on the early events in Fas apoptotic signaling. (A) Gadd45β does not affect DISC formation. DISC analysis with lysates from Neo, HA-Gadd45β (Gadd45β), Bcl-x_L, and DN-FADD BJAB clones treated with anti–APO-1 for the times indicated. Western blots were performed using anti–APO-1 immunoprecipitates (left panels) or total cell lysates (right panels) and antibodies against caspase-8, FADD, or Fas, as indicated. Fas served as loading control. Note that in the particular BJAB clone shown, DN-FADD inhibited Fas-induced recruitment of endogenous FADD. Specific and nonspecific (n.s.) bands are labeled. Shown is one representative of 3 independent experiments. (B) DISC-associated caspase-8 activity in untreated (–) and anti–APO-1–treated (30 minutes; +) BJAB clones. Clones, Fas stimulation, and DISC immunoprecipitations were as in panel A. Caspase-8–specific activity was measured by fluorimetric assays using zIETD-AFC as in Figure 4B. Values show absolute fluorescence units and represent the mean ± standard deviation of 3 independent experiments. (C) Gadd45β has no effect on the direct Fas-induced processing of Bid. Western blots (50 μg) showing Bid cleavage in Neo and HA-Gadd45β BJAB clones transduced with pBabe-Bid and treated with anti–APO-1 for the times indicated. pBabe-transduced cells were left untreated. Antibodies and Bid-specific bands are labeled.