Rabbit anti histone h3 trimethyl k4 antibody

The antibodies used for western blot analysis were rabbit anti-SET1 polyclonal antibody (1:1,000, BETHYL, A300–289A), rabbit anti-BTG2 polyclonal antibody (1:2,000, GenWay Biotech, GWB-D54FE7), rabbit anti-Histone H3 antibody (1:1,000, Abcam, ab1791), rabbit anti-Histone H3 monomethyl K4 antibody (1:500, Abcam, ab8895), Goat anti-Histone H3 dimethyl K4 antibody (1:1,000, Abcam, ab11946), rabbit anti-Histone H3 trimethyl K4 antibody (1:1,000, Abcam, ab8580) and mouse anti-Actin monoclonal antibody (1:5,000, BD Bioscience, 612656). Cells were lysed in RIPA buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10 mM NaF, 0.1% SDS, 1% Nonidet P-40 and 1 × protease inhibitor mixture (Roche)), proteins were separated on a 4–15% polyacrylamide gradient–SDS gel (Bio-Rad), transferred on polyvinylidene difluoride membranes (Millipore) and blocked in TBST (Tris-buffered saline and Tween 20; 25 mM Tris, pH 7.4, 136 mM NaCl, 5 mM KCl and 0.1% Tween) containing 5% milk. The antibodies were used at each dilution, as described above, in 1% milk/TBST before use. Blots were incubated with the primary antibodies for 2 h at room temperature. Blots were washed (three times) with 1% milk/TBST and were incubated with the appropriate horseradish peroxidase-conjugated antibodies. Bound antibodies were detected with enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech).

Total cell proteins were extracted using RIPA lysis buffer (Beyotime). The protein concentration of each sample was measured using Enhanced BCA Protein Assay Kit (Beyotime). 30 μg was taken from each sample for electrophoresis in 10% SDS‐PAGE gels, the separated proteins in gels were transferred to polyvinyl difluoride membranes, which was then blocked with QuikBlock Blocking Buffer for 15‐30 minutes (Beyotime). After that, the membranes were separately incubated with rabbit anti‐MLL1 antibody (dilution 1:1000; US Biological), mouse anti‐HOXA10 antibody (dilution 1:500; Santa Cruz biotechnology), rabbit anti‐Histone H3 (tri‐methyl K4) antibody (dilution 1:1000; abcam), rabbit anti‐PGR antibody (dilution 1:1000; CST), mouse anti‐β‐actin antibody(dilution 1:6000; OriGene) and mouse anti‐GAPDH (dilution 1:6000; Proteintech) overnight at 4°C. On the second day, the membranes were washed with PBS and then incubated with the peroxidase‐conjugated second antibody for 1 hour at room temperature. After washing again with PBS, membranes were visualized by ECL system (Tanon, Shanghai) using the High sensitivity ECL chemiluminescence detection kit (Vazyme, China). Image J was used for semi‐quantitative analysis of protein expression.