Sc 56070

Western Blot analysis was performed as described before^{35 (link)}. In brief, cells were homogenized in RIPA lysis buffer supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). Protein lysates were resolved on SDS-PAGE gels before being transferred to the PVDF membrane. Anti-FADD, anti-Caspase-8, anti-RIP, and anti-RIP3 antibodies were purchased from Santa Cruz (sc-271748, sc-56070, sc-133102, and sc-374639 respectively). Mouse monoclonal anti-Actin antibody was used as a loading control. The densities of bands were quantitated using ImageJ software.

WSU12 cells, treated with vehicle control (DMSO) or YOK1104 for 12 h in the presence or absence of XRT (6 Gy), were lysed in binding buffer [20 mM Tris-HCl, pH 7.6, 125 mM NaCl, and 1% Nonidet P-40 with a cocktail of protease and phosphatase inhibitors (Roche)] through one cycle of freezing in liquid nitrogen and thawing in a 42 °C water bath. Genomic DNA was sheared by passing the extracts through a 26-gauge needle four times. The resulting lysates were centrifuged at 3000 × g for 15 min to pellet the cellular debris. Total 500 μg proteins were incubated with 2 μg control IgG or mouse monoclonal antibody raised against full-length recombinant caspase-8 of human origin (Santa Cruz, sc-56070) for overnight, followed by incubation with 40 μl of 50% protein A agarose bead slurry for 1 h. The beads were washed with the binding buffer for 5 min at 4 °C with gentle agitation; washing was repeated three times. The proteins bound to protein A/G agarose beads were dissociated in 2X SDS sample buffer, heated at 100 °C for 5 min, and separated on a SDS-PAGE. For immunoblot analysis of caspase-8 using caspase-8 IP products, the secondary HRP antibody (GeneTex, GTX221667-01), which specifically reacts with the native, non-reduced form of mouse IgG, was used to distinguish the caspase-8 band from immunoglobulin heavy chain bands.