Chemiluminescent ecl reagent

Fresh tissues and cells were lysed with cell lysis buffer (Beyotime Biotechnology) and western blot was performed as described previously [18 (link)]. Briefly, 40 μg total proteins were applied to separation with SDS–PAGE gel. After the electrophoresis, the proteins were transferred to PVDF membranes (Millipore), followed by blocking in the TBST buffer containing 5% fat-free milk. The membranes were then incubated with indicated antibodies overnight at 4 °C, and then washed and incubated with HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 h at room temperature and finally visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following antibodies were used in this work: Anti-GAPDH (Cell Signaling Technology), anti-JMJD2A (Cell Signaling Technology), anti-Histone H3 (Santa Cruz Biotechnology), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-mTOR (Cell Signaling Technology), anti-p-mTOR (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt Thr308 (Cell Signaling Technology), anti-S6K1 (Cell Signaling Technology), anti-p-S6K1 (Cell Signaling Technology).

Immunoprecipitation analysis was performed using standard protocols based on previously described methods22 (link). Cultured cells were lysed with Cell Lysis Buffer (Beyotime) supplemented with a protease inhibitor cocktail (Roche). A total of 40 μg protein were separated on a 12% SDS–polyacrylamide gel (SDS-PAGE). After electrophoresis, the proteins were transferred to PVDF membranes, followed by antigen-blocking in 5% fat-free milk. The membranes were then probed with the indicated antibodies overnight at 4 °C, and then washed and incubated with primary-antibody-matched and HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 hours. Finally, the membranes were washed and visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following primary antibodies were used: anti-Myc antibody (Santa Cruz Biotechnology), anti-GST antibody (Santa Cruz Biotechnology), anti-SIRT1 antibody (Santa Cruz Biotechnology), anti-Nkx2.5 antibody (Santa Cruz Biotechnology), anti-HA antibody (Santa Cruz Biotechnology), anti-Flag antibody (Sigma), anti-acetylated lysine (ac-K) antibody (Cell Signaling Technology), and anti-actin antibody (Santa Cruz Biotechnology).