After incubation, the cells were harvested and lysed in ice-cold 1% NP-40 lysis buffer containing 150 mM NaCl, 10 mM HEPES (pH 7.45), 5 mM sodium pyrophosphate, 5 mM sodium fluoride, 2 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Applied Science). Whole cell extracts were obtained from the supernatants after centrifugation of the cell lysate at 13,000 × g for 15 min at 4°C. Protein concentration was quantitated using the Bradford assay. Proteins (50 µg) were separated via SDS-polyacrylamide gel electrophoresis (PAGE; 8–13%) and transferred onto 0.45 µM nitrocellulose membranes (Amersham; Cytiva). The membranes were blocked for 60 min at room temperature using 5% (w/v) BD Difco skim milk powder (Thermo Fisher Scientific, Inc.) and immunoblotted with primary antibodies at 4°C overnight at optimal dilution (Table I). After washing three with Tris-buffered saline with 0.1% Tween 20 buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:2,000 for 1 h at room temperature. Immunoreactivity was detected using ECL Plus (Amersham; Cytiva) and digitalized using Image Quant LAS 4000 (GE Healthcare).