Figure 5.

Induction of ligand-induced receptor-bound CD95 complex (DISC) or intracellular caspase-8–containing complex (complex II) in the presence or absence of the IAP antagonist. (A) The CD95 DISC was precipitated from MET1 or A5RT3 cells preincubated with 10 µM zVAD-fmk and 100 nM of the IAP antagonist for 1 h and subsequently treated with CD95L-Fc for 2 h. CD95L DISC (left) was precipitated as detailed in Materials and methods. Precipitation of receptor complexes after lysis (−) served as internal specificity control when compared with ligand affinity precipitates (IP; +). Equal amounts of DISC (CD95L IP) or complex II (caspase-8 IP) were subsequently analyzed by Western blotting for the indicated molecules. Equal amounts of total cellular lysates (TL) were loaded on the same gels to allow comparison of signal strength between CD95L-IP, complex II, and total cellular lysates. (B) Kinetics of DISC (left) or complex II (right) in the presence or absence of the IAP antagonist. The CD95 DISC was precipitated from parental HaCaT cells either prestimulated with 100 nM of the IAP antagonist and 10 µM zVAD-fmk for 1 h alone or the combination of both and subsequently stimulated with 250 U/ml CD95L for the indicated times. CD95L DISC (left) or complex II (right) was precipitated as detailed in Materials and methods and specified for A. MM, molecular mass.