Figure 4

DR4 is redistributed to lipid raft subdomains in R1 cells. (a) Western blot analysis for various proteins important in regulating the extrinsic apoptotic pathway. GAPDH was used as loading control. (b) R1 cells were preincubated with soluble monoclonal antibodies against DR4 or DR5 (μg) for 1 h followed by 24 h of treatment with 50 ng/ml of TRAIL. Cell viability was determined by MTT assay and expressed as % of untreated control. (c) R1 cells were transiently transfected with either scrambled siRNA or siRNA against DR4 or DR5 for 48 h followed by 50 ng/ml of TRAIL treatment for 24 h and cell viability were determined using MTT assay; *P<0.05. (d) R1 cells were preincubated with blocking antibodies against DR4 and DR5 for 1 h followed by 24 h of treatment with 50 ng/ml of TRAIL and lysates were subjected to western blot analysis for the assessment of caspase 3 and caspase 8 processing as well as PARP cleavage. (e) WT (top) and R1 cells (bottom) were treated with 50 ng/ml TRAIL for 15 min and subjected to discontinuous sucrose density gradients of Triton X-100 cell lysates for separation of lipid raft and non-raft fractions. One to 9 fractions were examined by western blots for the presence of DR4 and DR5 (first two rows). Lipid raft fractions 5 and 6 were identified by western blots. Flotillin and caveolin-1 were used as lipid raft markers. (f) Following TRAIL (50 ng/ml) treatment for 15 min, WT and R1 cells were subjected to discontinuous sucrose density gradients of Triton X-100 cell lysates for separation of lipid raft and nonraft fractions. Fractions 4–6 were collected and immunoprecipitated with anti-caveolin-1 antibody. As shown, caveolin-1 and FADD protein expression were detected by immunoblotting. (g) R1 cells were preincubated with the indicated doses of MCD for 1 h followed by 24 h of treatment with 50 ng/ml of TRAIL. Whole-cell lysates were subjected to western blot analysis for the assessment of caspase 3 and caspase 8 processing as well as PARP cleavage. GAPDH was used as loading control. Data shown are mean±S.D. of at least three independent experiments