Anti jmjd2a

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).

Cells were lysed in lysis buffer containing 50 mM Tris–HCl pH6.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate and 1% NP-40 and 1 × complete protease inhibitor cocktail (Roche), 1 μg/ml antipain, 1 μg/ml leupeptin, 1 mg/ml pepstatin, 2 μg/ ml aprotinin, 1 mM PMSF on ice for 30 min. The cell lysates were loaded on a 8–10% SDS–polyacrylamide gel for SDS-PAGE, followed by electro-transfer to a PVDF membrane. The membrane was blocked with 5% skim milk in TBS-Tween-20 (0.1%, v/v) for 2 h and then incubated with either anti-GAPDH (Cell Signaling Technology, MA, USA, #2118, 1:1000), anti-JMJD2A (Cell Signaling Technology #5328, 1:1000), anti-JMJD2B (Abcam ab191434, 1:1000), or anti-JMJD2C (Novus Biologicals, CO, USA, NB110-38884, 1:1000) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody. Immunoreactive protein was visualized using ECL prime solution (GE Healthcare, IL, USA).