Preparation of cell lysates, protein gel electrophoresis, and immunoblotting

Cells grown on tissue culture plates were washed with 1X PBS and prepared in cold Triton lysis buffer (1% Triton X-100, 40 mM HEPES (pH 7.5), 120 mM sodium chloride, 1 mM EDTA, 1 mM phenyl methylsulfonyl fluoride, 10 mM sodium pyrophosphate, 1 μg/ml each of cymostatin, leupeptin and pepstatin, 10 μg/ml each of aprotinin and benzamidine, 2 μg/ml antipain, 1 mM sodium orthovanadate, 50 mM sodium fluoride). For immunoblotting, cell lysates were centrifuged at 14,000 rpm for 3 min at 4°C to remove cell debris. Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad) with bovine serum albumin (RMBIO) as standard then were prepared in Laemmli SDS-PAGE sample buffer. Aliquots of protein lysate were resolved by SDS-PAGE, transferred to nitrocellulose membranes (0.45 μm) (Bio-Rad, Hercules, CA) and immunoblotted with the indicated antibodies at 1:1000 dilution, followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Sigma-Aldrich) and Amersham ECL Select Western Blotting Detection Reagent or Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Hudson, NH). Signals were detected using ChemiDoc Touch Imager (Bio-Rad) or CL1500 Imaging System (Thermo Fisher Scientific). For western blot signal quantitation, the Image Lab software (Bio-Rad) was used.