Phospho cdk2

Cells were sonicated (1 sec on and 1 sec off at 20% amplitude for 20 sec) in mammalian cell lysis buffer (20 mM HEPES, pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 10% glycerol) containing both protease and phosphatase inhibitors. Mouse skeletal muscle tissues were homogenized in RIPA buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS) containing both protease and phosphatase inhibitors. Lysates were cleared by centrifugation at 14,000 x g for 20 min, and protein amounts in the supernatant measured using the BCA (Pierce Biotechnology Inc., Rockford, IL, USA) assay. The resulting supernatant fractions were subjected to SDS-PAGE followed by western blot. Antibodies against Actinin, ERK1/2, ERK2, Myogenin, MyHC, Troponin C and β-Actin were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) while the ITPR1 antibody was from ABCAm (Cambridge, MA, USA). Antibodies against phospho-CDK2, phospho-EGFR, and phospho-ERK1/2 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). The antibody against GAPDH was developed in our laboratory.

Protein was extracted from the cells with RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Cat# 78 440, Thermo Fisher Scientific). The protein concentration was measured by Pierce BCA Protein Assay kit (Cat# 23 225, Thermo Fisher Scientific). Quantified protein lysates were resolved on SDS–PAGE gels, then transferred to polyvinylidene difluoride membranes. The primary antibodies against POU4F1 (Cat# ab81213 or Cat# ab245230, abcam), ERα (Cat# 8644, Cell Signaling Technology), Phospho‐Rb (Cat# 9308, Cell Signaling Technology), E2F2 (Cat# ab138515, abcam), CCND1 (Cat# 2978, Cell Signaling Technology), CDK2 (Cat# ab32147, abcam), Phospho‐CDK2 (Thr160)(Cat# 2561, Cell Signaling Technology), EZH2 (Cat# 5246, Cell Signaling Technology), PR (Cat# 8757, Cell Signaling Technology), H3K27me3 (Cat# 9733, Cell Signaling Technology), Phospho‐EZH2 (Thr416)(AF3585, Affinity Biosciences), GAPDH (Cat# 5174, Cell Signaling Technology) were used. HRP‐conjugated secondary anti‐rabbit or mouse antibody (Cat# 7074 or Cat# 7076, Cell Signaling Technology) was used and the antigen–antibody reaction was visualized using an enhanced chemiluminescence assay (Cat# 34 577, Thermo Fisher Scientific).