Anti jmjd2b

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).

Cells were lysed in ice-cold RIPA buffer (Abcam) containing protease inhibitor cocktail tablets (Sigma). Whole cell lysate protein concentrations were quantified using the standard Bradford method (Bio-Rad). Lysate aliquots containing 30-50 μg of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was then blocked by 5% skimmed milk powder for 1 h at room temperature, washed with TBST, and reacted with primary immunoglobulin G (IgG) unlabeled primary antibodies; anti-JMJD2B (Abcam), various modified and total histones (Cell signaling), β-actin (Sigma), at 1 : 1000 dilution overnight at 4°C. The secondary (anti-mouse and anti-rabbit) antibodies (Cell Signaling) were then reacted with the membrane at 1 : 1000 dilution for 1 h at room temperature. Chemiluminescence was detected using the ECL kit (Thermo Scientific Pierce). Protein band quantification was carried out using the Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad). β-Actin was used as a normalization control.