Western Blot Analysis of Cellular Proteins

Western blot analysis was performed as described previously [13 (link)]. The total protein from cell lines and tissues were extracted using RIPA lysis buffer (Beyotime, Shanghai, China) complemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China). The nuclear and cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Protein concentrations of cellular or nuclear extracts were determined using BCA Assay Kit (Thermo Fisher Scientifc, Waltham, MA, USA). The detail information of antibodies used in the present study were listed in Additional file 1: Table S5. β-actin was used as loading control. The proteins were visualized using enhanced chemiluminescence Western blot detection system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Then proteins were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (ECL, Thermo) and detected by a ChemiDocXRS system (BioRad). All of the experiments were performed three times independently.