Cell pellets were suspended in five volumes of lysis buffer (PBS containing 0.1% Triton X-100 and protease inhibitor cocktail) and sonicated on ice with two 10 s pulses (30 s pulse between two pulses). Cellular debris was removed by centrifugation (800 g, 10 min, 4°C) and the protein concentration of the supernatants quantified with a BCA protein assay kit (Thermo Scientific) with bovine serum albumin as a standard.
For Western blotting, protein samples (50 µg) were subjected to reducing and denaturing SDS-PAGE (4–15% Criterion™ Tris-HCl gel, Bio-Rad, Hercules, CA) followed by electroblotting onto nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk in PBST (PBS containing 0.1% Tween 20) for 30 min at 37 °C. The blots were reacted with primary antibodies overnight at 4°C, and then with a secondary antibody conjugated to HRP (Bio-Rad) for 30 min at 37 °C. Specific proteins were visualized using chemiluminescence.
For Western blotting, protein samples (50 µg) were subjected to reducing and denaturing SDS-PAGE (4–15% Criterion™ Tris-HCl gel, Bio-Rad, Hercules, CA) followed by electroblotting onto nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk in PBST (PBS containing 0.1% Tween 20) for 30 min at 37 °C. The blots were reacted with primary antibodies overnight at 4°C, and then with a secondary antibody conjugated to HRP (Bio-Rad) for 30 min at 37 °C. Specific proteins were visualized using chemiluminescence.