Sc 6246

The MESCs were lysed, homogenized in 1% SDS lysis buffer supplemented with Halt protease inhibitor cocktail (Thermo Scientific) and kept for 10 min at 95°C. Lysates were centrifuged at 1800 x g for 5 min to remove cell debris. Protein content of the lysate was estimated using the Pierce BCA protein assay kit (ThermoFisher Scientific). Equal amounts of protein lysates were separated by SDS-PAGE gel electrophoresis (Bio-Rad) and transferred to Immobilon-P PVDF membrane (Millipore). Non-specific binding was blocked using 5% nonfat dry milk for 1 h at room temperature and then the membrane was incubated with a primary antibody against p21 (Santa Cruz Biotechnology sc-6246, 1:250), p16 (Cell Signaling Technology #80772, 1:1000), p53 (Cell Signaling Technology #2524, 1:1000), H3K27me3 (Cell Signaling Technology #9733, 1:1000) p-AKT or AKT (Cell Signaling Technology #4060, 1:2000 or #4685, 1:1000, respectively) or GAPDH (Proteintech 60004-1-Ig, 1:5000) at 4°C overnight. The next day, membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for 1h. Antibody binding was visualized with SuperSignal west pico PLUS chemiluminescent substrate (Thermo Scientific) and images were captured by C-DiGit blot scanner (LI-COR Biosciences). Band intensities were quantified with ImageJ software.