Recruitment of death receptors and downstream signaling molecules and DISC formation in membrane rafts following ALP treatment of MM cells. (A) Untreated MM144 cells (control) and MM144 cells treated with 10 μM edelfosine or perifosine for 15 hours were lysed in 1% Triton X-100 and fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was subjected to SDS-PAGE before analysis of the indicated proteins using specific antibodies. The migration positions of the 55-kDa procaspase-8 as well as of the cleavage products (arrows) are denoted. Location of GM1-containing lipid rafts (fractions 4-6) was determined using CTx B subunit conjugated to horseradish peroxidase. (B) Fas/CD95 was immunoprecipitated from untreated control or edelfosine-treated (15 hours) MM144 cell extracts. Fas/CD95 was also immunoprecipitated from MM144 cells pretreated with MCD to disrupt lipid rafts and then treated for 15 hours with edelfosine. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with specific antibodies against Fas/CD95, FADD, and caspase-8. (C) Fas/CD95 was immunoprecipitated from a pool of membrane raft–enriched fractions 4 to 6 from sucrose gradients similar to those shown in Figure 4A of edelfosine-treated MM144 cells. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with Fas/CD95-, FADD-, and caspase-8–specific antibodies, respectively. Membrane raft–enriched fractions were also immunoprecipitated with P3X63 (X63) myeloma supernatant as a negative control, rendering no signal. Experiments shown are representative of 3 performed.