Anti procaspase 8

LNCaP cells were adhered in Petri dishes (3.5 cm diameter) for 48 h and then treated with 100 ng/mL TRAIL and/or 25 µM xanthohumol. The cells were washed twice with cold PBS and lysed in a Ripa Buffer with Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein samples (20 µg) were separated on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels using a Mini Trans-Blot (BioRad, Hercules, CA, USA). Proteins were transferred onto a Whatman nitrocellulose membrane (Labo Plus, Warsaw, Poland). The membrane was blocked in 5% non-fat dry milk for 1 h in room temperature and incubated at 4 °C overnight with the following primary antibodies (1:1000 dilutions): anti-procaspase 3, anti-procaspase 8, anti-procaspase 9, anti-Bid, anti-cytochrome c, anti-Bax, anti-Bad, anti-Bcl-2, anti-Bcl-xL, and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA). After thorough washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Then the membranes were incubated for 1 min with a chemiluminescence detection chromogen Phototope^®-HRP Western Blot Detection System (Cell Signaling Technology). The detection of chemiluminescence and photographic documentation was performed using ChemiDoc™ XRS+ Systems with Image Lab™ software (BioRad).

Antibodies; Rat-anti mRIPK3 and rat anti-mMLKL 8F6 (selected for affinity to residues 1–30 of mouse MLKL) and rat anti-MLKL 3H1^{4 (link)} (MLKL brace region) were produced in-house. Anti-Pro Caspase 8 (#4927) and GAPDH (#2113) were purchased from Cell Signaling Technology. Anti-mouse MLKL pS345 (ab196436) and anti-Actin (ab5694) were purchased from Abcam. Anti-VDAC (AB10527) was purchased from Millipore. Fc-hTNF was produced in house and used at a final concentration of 100 ng per mL. Recombinant mouse IFN-γ and β were purchased from R&D Systems (Minneapolis, MN, USA) Q-VD-OPh and zVAD-fmk were purchased from MP Biomedicals (Seven Hills, NSW, Australia). Smac mimetic also known as Compound A, and the caspase inhibitor IDN-6556 were a gift from TetraLogic (Malvern, PA, USA). Propidium iodide, doxycycline, and bafilomycin were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).