Proteinase inhibitor cocktail and phosphatase inhibitor cocktail

To isolate total proteins, cells were washed with cold PBS and resuspended in lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1.0 mM EDTA, pH 7.5, 10% glycerol, 1 mM BME, 1% NP40) plus proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Penzberg, Upper Bavaria, Germany). After incubation for 30 min. on ice, the supernatant was collected by centrifugation at 12,000 × g for 15 min. at 4°C, and the protein concentration was determined by the Bradford method. Sample containing equal proteins (40 μg) were loaded and analysed by immunoblotting. Briefly, proteins were separated by 12% SDS‐PAGE and transferred onto PVDF membrane (Life Technologies). Membrane were blocked with blocking buffer (5% non‐fat dry milk, 20 mM Tris‐HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for at least 1 hr at room temperature. Membranes were incubated with primary antibodies in the above solution on an orbit shaker at 4°C overnight. Following primary antibody incubations, membranes were incubated with horseradish peroxidase‐linked secondary antibodies (anti‐rabbit, antimouse or anti‐goat IgG). Antibody‐bound protein bands were detected using high sensitive Immobilon Western Chemiluminescent HRP Substrate (Millipore), and photographed with Bio‐Rad Chemiluminescence Imaging System (Bio‐Rad Laboratories, Inc. Hercules, CA, USA).

For isolation of total proteins, cells were rinsed with cold PBS and resuspended in lysis buffer (0.5 M NaCl, 50 mM Tris, 10% glycerol, 1.0 mM EDTA, 1mM BME, 1% NP40, plus proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). The supernatant was collected by centrifugation at 12,000 g for 15 min at 4 °C. The concentration of isolated protein was determined by the Bradford method. Isolated proteins (40 μg) were loaded and analyzed by immunoblotting. Membranes with transferred protein were incubated with primary antibodies in the blocking buffer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20 on an orbital shaker at 4 °C overnight. Membranes then were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG). Antibody-bound protein bands were detected using high sensitive Immoblot Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA), and photographed with Bio-Rad Chemiluminescence Imaging System (Bio-Rad Laboratories, Inc. Hercules, CA, USA)