Figure 7.
cFLIPL but not cFLIPS blocks the formation of complex II. (A) DISC or complex II formation in the presence or absence of the IAP antagonist. To allow for caspase activity, these experiments were performed without zVAD-fmk. HaCaT cells were stimulated with CD95L-Fc for 2 h. Subsequently, the CD95L DISC (left) was precipitated using ligand affinity precipitation as detailed in Materials and methods. Precipitation of receptor complexes after lysis (−) served as an 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 IP and total cellular lysates. (B) DISC or complex II formation in parental HaCaT. Cells were either pre- or co-stimulated with 10 µM zVAD-fmk, 50 µM Necrostatin-1, and 100 nM of the IAP antagonist for 1 h and subsequently stimulated with 250 U/ml CD95L for 2 h. The CD95L DISC (left) or complex II (right) was precipitated as detailed in Materials and methods and specified for A. Equal amounts of DISC (CD95L IP) or caspase-8–interacting proteins (complex II) were subsequently analyzed by Western blotting for the indicated molecules. MM, molecular mass.