Pierce enhanced chemiluminscent

Cells were lysed with NP-40 buffer (1% NP-40, 0.15 M NaCl, 50 mM Tris, pH 8.0) containing phosphatase inhibitors (okadaic acid; catalog no. ab120375; Abcam). Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis on 12% gels and transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing Tween (TBST) for 1 h, membranes were incubated with primary antibody in TBST overnight at 4°C and subsequently incubated with anti-mouse (catalog no. A9309; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) or anti-rabbit (catalog no. ab97051; Abcam) horse radish peroxidase-conjugated secondary antibodies at a 1:2,000 dilution for 2 h at room temperature. Reactive proteins were detected using Pierce Enhanced Chemiluminscent and SuperSignal™ Chemiluminescent substrates (Thermo Fisher Scientific, Inc., Waltham, MA, USA). To control for loading efficiency, the blots were stripped with Mild Stripping Buffer (Abcam) at room temperature for 5–10 min and reprobed with GAPDH antibody (1:600).