Comprehensive Western Blotting Protocol

Western blotting was carried out as previously described 27 (link)-29 (link). The protein extraction buffer (Beyotime, Shanghai, China) or nucleoprotein extraction kit (Sangon Biotech, C500009) was used to extract total or nuclear cellular proteins according to the manufacturer's instructions. Further, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk or 5% Bovine Serum Albumin, the membrane was probed with the following primary antibodies: anti-β-actin (1:2000), anti-Histone H3 (1:1000), anti-MSR1 (1:1000), anti-AKT (1:1000), anti-p-AKT (1:1000), anti-GSK3β (1:1000), anti-p-GSK3β (1:1000), anti-mTOR (1:1000), anti-p-mTOR (1:1000), and anti-β-catenin (1:1000). The species-matched secondary antibodies were used (1:10000), and the bands were detected by the Odyssey imaging system (LI-COR, Lincoln, NE, USA).