Fig. 4. cFLIP cleavage counteracts complex-II formation.

(A) Cflip^WT and Cflip^D377A MDFs were treated with TNF (10 ng/ml), Smac mimetic (250 nM), and emricasan (1 μg/ml) for 4 hours, and lysates were separated on a Superose 6 size exclusion column. Aliquots from each fraction were retained and analyzed by immunoblotting with the indicated specific antibodies. (B) Cartoon depicting cFLIP domain composition and position of the D377 and Q469 residues (left). Immunoblotting analysis of Cflip^−/− MDFs reconstituted with the indicated cFLIP constructs via lentiviral infection or with an empty lentivirus (EV). cFLIP- and tubulin-specific antibodies were used (right). (C and D) Cflip^−/− MDFs reconstituted as in (B) were treated with TNF (10 ng/ml) (C) or TNF (1 ng/ml) and emricasan (1 μg/ml) (D) and cell death was measured over time by calculating the percentage of Sytox Green–positive cells (n = 3). (E) MDFs Cflip^−/− MDFs reconstituted as in (C) and treated as in (D) for 3 hours. Cell lysates were analyzed by immunoblotting with the indicated specific antibodies.