Western Blotting

2 × 2 × 2 mm³ tumor tissue explants were lysed group wise with 50 mM HEPES (pH 7.4), 100 mMNaCl, 0.1% CHAPS, 1 mM DTT, and 0.1 mM EDTA and protease inhibitor cocktail (Calbiochem) containing buffer to extract the total cellular protein. Protein concentration was determined by Bradford assay (BIO-RAD, Protein Assay, cat no-500-0006). 30 μg of total cellular protein from each group were electrophoresed on 10% SDS–polyacrylamide gels followed by their transfer onto nitrocellulose membranes. The membranes were blocked with 10% skimmed milk in Tris Buffered Saline with Tween 20 (TBST) for 2 h at room temperature, followed by overnight incubation at 4°C with p-AKT (Cell signaling Technology, cat no-4060), p-ERK and β-actin (MP Biomedicals, cat no-0869100) diluted 1:1,000 in 1% BSA in TBST. Blots are then washed with TBST followed by incubation with Goat Anti-Rabbit IgG Polyclonal Antibody (HRP) (KPL) and Peroxidase AffiniPure Rabbit Anti-Mouse IgG (H + L) (Jackson ImmunoResearch LABORATORIES, INC.) at a dilution of 1:5,000 in TBST. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific™) was used for developing blots and the images were taken using XRS+ imaging system (Bio-Rad). Densitometry analysis of the blots was done using ImageJ software. Values of p-AKT and p-ERK were represented normalized to β-actin (33).