Protein Extraction and Western Blotting Procedure

Whole cell lysates were prepared by lysing cells with RIPA buffer (0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 25 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor mixture and PhosSTOP (Roche). Histones were prepared by acid extraction as previously described (Choi et al., 2016 (link)). Proteins were quantified with Bradford assay (Bio-Rad). Equal amounts of proteins were separated with SDS-PAGE and transferred to nitrocellulose membranes. To visualize equal protein loading, blots were stained with Ponceau S. Blots were incubated in 5% non-fat milk in TBST, probed with primary antibodies to HA (1:1,000, Cell Signaling Technology, Cat# 2367), CtBP2 (1:1000, BD Biosciences, Cat# 612044, RRID:AB_399431), Mek1/2 (1:1,000, Cell Signaling Technology, Cat# 9122), alpha-tubulin (Sigma, Cat# T5168), MLL1 (1:1,000, Cell Signaling Technology, Cat# 14197), CtBP1 (1:1,000, BD Biosciences, Cat# 612042, RRID:AB_399431), Myc tag (1:1,000, Cell Signaling Technology, Cat# 2276), V5 (1:1,000, Invitrogen, Cat# R950-25), Histone H3K4me3 (1:1,000, Cell Signaling Technology, Cat# 9751, RRID:AB_2616028), and Histone 3 (1:1,000, Cell Signaling Technology, Cat# 5427) and then were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by enhanced chemical luminescence (Cytiva or Pierce).