Western blotting analysis

Western blotting analysis was performed as previously described⁶⁹. Briefly, tumor cells were cultured in the presence of the indicated ceramide analogs or ceramide analogs plus MegaFasL for 4 h. Cells were collected and lysed in cytosol buffer [10 mM Hepes, pH 7.4, 250 mM Sucrose, 70 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitor cocktails (Calbiochem, Billerica, MA), and 0.01% digitonin] for 10 min. Cytosolic fractions were resolved in 4–20% SDS polyacrylamide gel and analyzed by Western blotting. Anti-cleaved caspase 8 and anti-cIAP1 were obtained from R&D Systems. Anti-cleaved human PARP, anti-xIAP, anti-p38, anti-p-p38, anti-pErk1/2, anti-Erk1/2, anti-pJNK, and anti-JNK antibodies obtained from Cell Signaling. β-actin was obtained from Sigma-Aldrich.