Anti chk1 ps317

Cells were collected and lysed with pre-cooled RIPA lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and fresh protease inhibitor cocktail. The cell lysate was centrifuged at 13,000 rpm/min at 4°C for 20 min and the supernatant was collected. After determining the protein concentration using the BCA Protein Assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to polyvinylidene fluoride membranes. After blocking with 5% (w/v) non-fat milk in Tris-buffered saline at room temperature for 1 h, the membranes were separately incubated with the following primary antibodies: anti-Nestin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Chk1 (Santa Cruz), anti-p53 (Santa Cruz), anti-Chk1 pS317 (Cell Signaling Technology, Danvers, MA, USA), and anti-β-tubulin (Boster Biological Technology, Wuhan, China) for 2 h at room temperature. After extensive washing with 0.1% Triton X-100 in Tris-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Boster Biological Technology) for 1 h at room temperature before analysis using electrochemiluminescence western blotting detection reagents.