Pierce ecl western blotting substrate

Cells were washed twice with ice-cold PBS and lysed with non-ionic denaturing lysis buffer (50 mM Tris, pH7.5, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet-P40, and 10% glycerol), containing protease and phosphatase inhibitor cocktail (Pierce). The cell lysates were vortexed vigorously and centrifuged at 16,000g for 15 min at 4°C. The protein concentration of the lysates was estimated using Bradford Ultra Protein Assay reagent (Expedeon). 40 μg proteins were resolved by SDS–PAGE on 10% gels. The resolved proteins were subsequently transferred onto polyvinyl difluoride (PVDF) membranes (Pierce). The membrane was blocked using 1% BSA in Tris-buffered saline in 1% Tween-20 (TBST). The blots were then incubated with antibodies at their optimal dilutions overnight at 4°C. The anti-rabbit or mouse HRP-conjugated secondary antibodies (Pierce) were diluted at 1:5,000 in blocking buffer. The blots were incubated with the secondary antibodies for 1 h at room temperature. Visualization was performed using the Pierce ECL Western Blotting substrate for whole cell lysate immunoblotting or Advansta Western Bright ECL HRP substrate for subcellular fractionations and post-immunoprecipitation immunoblotting.