Human tissues for Western blotting were lysed in RIPA (50 mM Tris pH 7.4, 1% NP40, 150 mM NaCl, 40 mM NaF, 1 mM Na3VO4, 1 mM EDTA and 10 μl/ml of protease inhibitor cocktail) buffer. For cell lines, the cells were seeded in dishes at 60% confluence overnight and then treated with emodin or vehicle control for 24 h. The cells were lysed in ice-cold RIPA buffer according to previously described methods [56 (link)]. Protein concentrations were quantified using a BCA assay (Thermo Scientific Pierce) according to the manufacturer's protocol. Lysate 5-40 μg was loaded into each lane by Western blotting. The specific signal was detected using a secondary antibody coupled with horseradish peroxidase (Jackson ImmunoResearch Labs) and Chemilucent Plus Western Blot Enhancing Kit (Millipore, Bedford, MA, USA).
The antibodies used included anti-EZH2 (Clone: AC-22, CellSignaling, Danvers, MA, USA), anti-Actin (Sigma, St Louis, MI, USA), anti-V5 tag (AbD Serotec, USA), anti-H3K27me3 (Merck-Millipore, Upstate, Lake Placid, NY, USA), anti-EED (Abcam, Cambridge, UK), anti-Suz12 (CellSignaling, Danvers, MA, USA), anti-HA (Clone:12CA5, Roche), and anti-GAPDH (Sigma, St Louis, MI, USA). Secondary antibodies, including goat-anti-mouse-HRP and goat-anti-rabbit-HRP, were purchased from Jackson ImmunoResearch Labs (Westgrove, CA, USA).
The antibodies used included anti-EZH2 (Clone: AC-22, CellSignaling, Danvers, MA, USA), anti-Actin (Sigma, St Louis, MI, USA), anti-V5 tag (AbD Serotec, USA), anti-H3K27me3 (Merck-Millipore, Upstate, Lake Placid, NY, USA), anti-EED (Abcam, Cambridge, UK), anti-Suz12 (CellSignaling, Danvers, MA, USA), anti-HA (Clone:12CA5, Roche), and anti-GAPDH (Sigma, St Louis, MI, USA). Secondary antibodies, including goat-anti-mouse-HRP and goat-anti-rabbit-HRP, were purchased from Jackson ImmunoResearch Labs (Westgrove, CA, USA).