Anti pikb

Cell pellets were suspended in 0.1 ml of ice-cold RIPA buffer and incubated on ice for 1 h. When subcellular fractions were prepared, the Subcellular Proteome Extraction Kit (Merck, Darmstadt, Germany) was used according to the manufacturer’s instructions. Protein concentrations were determined by DC protein assay (Bio-Rad, Hercules, California, USA) and equivalent amounts of total cellular protein were separated by 3–8% or 4–12% gradient gels. The proteins were electrophoresed using a polyacrylamide gel and the resolved proteins were transferred to a polyvinylidene fluoride membrane and detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) after a specific antibody reaction. Anti-β-actin and anti-Mcl-1 antibodies were purchased from Sigma-Aldrich and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. Anti-p-IkB, anti-NF-κB, anti-Bcl-2, anti-Bcl-xL, anti-p-p38, anti-cleaved caspase-3, and anti-Lamin A/C antibodies were purchased from Cell Signaling Technology. Results were analyzed and quantified using a CS Analizer 3 (Atto Corporation, Tokyo, Japan).

Cell protein extracts were analyzed by western blotting, as previously described [12 (link)]. The following primary antibodies were used: anti-LIGHT and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pAKT, anti-pIkB, anti-pJNK, anti-total AKT, anti-IkB, anti-total-ERK and anti-total JNK (Cell Signaling, San Diego, CA, USA).

CPT was purchased from Selleckchem company (Houston, TX, USA). Anti-STAT3, anti-p-STAT3 (705), anti-AKT, anti-p-AKT (473), anti-IKB, and anti-p-IKB (32) were from Cell Signaling Technologies (Danvers, MA, USA). Anti-GAPDH was from KangChen company (Shanghai, China).