Figure 6.
Caspase Activity in the Ripoptosome Is Differentially Regulated by cFLIP Isoforms and Is Negatively Regulated by Proteasome Function
(A and C) Ripoptosome formation was studied by caspase-8 IP.
(A) cFLIP isoforms differentially regulate caspase activity in the Ripoptosome. HaCaT-cFLIPS cells were stimulated with IAP antagonist and zVAD for 1 hr with poly(I:C) or diluent alone.
(B) In the absence of cIAPs, cFLIPS but not cFLIPL blocks caspase-8 activity in the Ripoptosome. HaCaT cells were transduced with retroviral vectors containing Flag-tagged cFLIPL or cFLIPS. Cells were prestimulated with IAP antagonist alone or subsequently with poly(I:C), followed by anti-Flag IP. Asterisks indicate nonspecific binding of Abs.
(C) Ripoptosome formation is negatively regulated by proteasome function in HaCaT-cFLIPS. Cells were prestimulated with MG132 for 10 min followed by stimulation with IAP antagonist for 4 hr. Experiments shown in (A)–(C) were performed in the presence of zVAD (10 μM).