Nfkbia

RIPA Lysis Buffer extracts proteins from cells and the concentration is measured by the BCA protein assay. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with particular primary antibodies (anti-Bcl-2; anti-Caspase-3; anti-SHPRH-146aa; anti-NFKBIA; anti-GAPDH) overnight at 4 °C after being blocked with 5% skim milk in TBST for 2 h at room temperature. These antibodies were used at Bcl-2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), Caspase-3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), NFKBIA (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:5,000; Cell Signaling Technology, Danvers, MA, USA). After thorough TBST washings, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:2,000; Cell Signaling Technology, Danvers, MA, USA) for an hour at room temperature, after which protein bands were unveiled via ECL technology.

Whole cell extracts from cultured cells were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail (Roche Diagnostics, Risch-Rotkreuz, Swizerland). Immunoblotting was carried out according to standard protocols with antibodies against TNFRSF9 (Sigma), LTA (GeneTex, San Antonio, TX), CYP1B1, CD27, IL8, THBS1 (Abcam, Cambridge, MA), SYK, FOS, MYC, NFKBIA, CASPASE-1 (Cell Signaling Technology, Danvers, MA). Antibody against GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Abcam) was used to confirm equal loading.