Pre cast mini gel

Cells were lysed in buffer containing 1% NP-40, 50 mM Tris, pH 7.5, 150 mM NaCl, and supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St Louis, MO, USA). After incubation on ice for 10 minutes, cell lysates were sonicated to ensure complete disruption. Lysates were then centrifuged for 10 minutes at 13,000 rpm and supernatants were subjected to protein quantification assay. For western blots, 50 μg of cell lysate was loaded on a pre-cast mini-gel (Bio-Rad, Hercules, CA, USA), followed by transferring to polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). For immunoprecipitation, cell lysates were diluted to 1 mg/ml with lysis buffer; 500 μg of total cell lysate was incubated with 2 μg of indicated antibody for 2 hours at 4°C, followed by addition of protein A/G Sepharose beads and further incubated for 1 hour at 4°C. After centrifugation at 1,500 rpm for 2 minutes, beads were washed three times with cell lysis buffer before analysis.