FIGURE 3.

TWEAK induces association of a death-signaling complex containing RIP1, FADD, and caspase-8, leading to caspase-8 activation. A, cells were treated with TWEAK (3 μg/ml) for the indicated time in the absence (D-dimethyl sulfoxide) or presence of zVAD-FMK and subjected to IP of caspase-8 followed by IB analysis of caspase-8 and RIP1. B, cells were treated with TWEAK (3 μg/ml, 6 h) and subjected to IP of RIP1 followed by IB analysis of FN14, FADD, caspase-8, or RIP1. C, cells were treated with TWEAK (3 μg/ml, 6 h), RIP1 was immunoprecipitated from cell lysates, and IPs were assayed for caspase-8 activity. D, cells were transfected with two siRNA oligonucleotides against RIP1 for 48 h, treated with TWEAK (3 μg/ml, 6 h) and subjected to IP of caspase-8 followed by IB analysis of FADD or caspase-8. E, cells were transfected with siRNA against FADD for 48 h, treated with TWEAK (3 μg/ml, 6 h), and subjected to IP of caspase-8 followed by IB analysis of FADD, caspase-8, or RIP1. F–H, cells were transfected with RIP1 siRNA as in D, treated with TWEAK for 48 h, and then analyzed for viability (F), caspase-3/7 activity (G), or MOMP (H). In F, IB analysis shows siRNA knockdown levels. For the IP experiments, zVAD-FMK (20 μm) was added 1 h prior to incubation with TWEAK. A vertical line denotes lane removal from the original image.