Western Blot Protein Detection Protocol

Western blots were performed as described in our previous study [9 (link)]. Cells were harvested at indicated time points and lysed for 1 h in lysis buffer (Santa Cruz) containing complete protease inhibitors (Roche Applied Science). Total protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit (Pierce, Thermo Scientific) after obtaining cell extracts by centrifugation at 13,000 rpm and 4 °C. Before transferred to PVDF membranes (Millipore), proteins were resolved on the sodium dodecyl sulfatesulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then the membranes were blocked in 5% non-fat-dry-milk solution for 1 h at room temperature and then blotted with specific primary antibodies over night at 4 °C, following incubated with horseradish peroxidase antibodies for 1 h at room temperature. The immunoreactive bands were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Thermo Scientific) or enhanced chemiluminescent substrate (ECL), followed by autoradiography.