For Western blot analyses, frozen cell pellets were rapidly re-suspended in ice-cold RIPA buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 0.5% Igepal-630, 0.5% deoxycholic acid, 0.1% SDS, 1 mM EDTA) supplemented with protease inhibitors (Complete Protease Inhibitor without EDTA, Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail A, Santa Cruz Biotechnology). One volume of 2× Laemmli buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 0.15% Bromophenol Blue, 20% glycerol, 200 mM β-mercaptoethanol) was added and the extracts were boiled for 5 min. Protein concentration for each sample was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amount of lysate for each sample was separated using SDS-PAGE and transferred to a nitrocellulose membrane. After transfer, the membrane was blocked in TBST (Tris-buffered saline and 0.1% Tween 20) supplemented with 5% milk and probed with the indicated primary antibody at 4°C overnight. After washing with TBST, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized using ECL reagents according
