FIG. 3.

The RASSF1A/TNF-R1/MOAP-1 complex migrates to distinct regions within a gel filtration (away from the DISC complex) and requires the presence of both MOAP-1 and RASSF1A. (A) U2OS cells were grown on 4- by 10-cm² dishes and left unstimulated or stimulated with TNF-α. Samples for gel filtration were treated as described in the legend to Fig. 2. Fractions were immunoblotted (IB) for endogenous DISC components: RIP1, FADD, TRADD, and caspase-8. These analyses were carried out two times. (B) The left panel shows annexin V staining of TNF-α-CHX-treated H1299 cells stably expressing control vector (−) or stably expressing Myc-MOAP-1 (+). The transfection efficiency of H1299 cells was between 30 and 50% GFP-positive cells. The experiment was repeated six times, and significance was evaluated by Student's t test (two tailed) with the indicated P value (*). The right panel shows expression of proteins used in annexin V staining.