Anti caspase 8 antibody

The TNFR complex I analysis was performed as described previously^{58 (link)}. Briefly, after a treatment with a series of HOIPIN-1 concentrations for 30 min, A549 cells were stimulated for the indicated times with 1 μg/ml FLAG-tagged TNF-α, and lysed in 1 ml lysis buffer, containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail, for 15 min on ice. The cell lysates were then immunoprecipitated with 15 μl anti-FLAG M2 beads (Sigma), overnight at 4 °C. For the TNFR complex II analysis, A549 cells were treated with 40 ng/ml TNF-α and 20 μg/ml CHX. HOIPIN-1 was added 30 min prior to the TNF-α- and CHX treatments. The cell lysates were then immunoprecipitated with an anti-caspase 8 antibody (Santa Cruz) and immunoblotted.

RPMI-1640 medium, fetal bovine serum, penicillin, streptomycin and trypsin were purchased from Gibco BRL (Grand Island, NY, USA). Atractylenolide I, MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] were purchased from Sigma (St. Louis, MO, USA). De-ionized water (ddH₂O) was collected from Millipore water purification system (Millipore, Milford, MA, USA). All rabbit polyclonal antibodies were purchased from Cell Signaling Technology (Dancers, MA, USA), except anti-caspase 8 antibody, which was purchased from Santa Cruz (Dallas, TX, USA).