Acetyl h3k27 d5e4

Cells were treated with DMSO or GSK126 for the indicated period of time and washed two times with 1× PBS. Cellular pellets were washed with buffer A (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM dithiothritol, and protease inhibitor cocktail) with 0.2% Triton X–100, and incubated on ice for 5 min. After centrifugation at 600 g, the nuclei were resuspended in buffer A without Triton X–100. Nuclei were then washed with buffer A without Triton X–100. Lysates were resuspended in Buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease inhibitor cocktail) and incubated on ice for 30 m. After centrifugation at 1,700g, at 4 °C, for 5 min, the nuclei were then washed with buffer B. Immunoblotting was performed. Antibodies to EZH2 (D2C9; 1:1,000), SUZ12 (D39F6; 1:1,000), H3 (9715; 1:6,000), Tri–methyl–Histone H3 Lys27 (C36B11; 1:1,000), Acetyl–H3K27 (D5E4; 1:1,000) antibodies were purchased from Cell Signaling. EED (05–1320, clone AA19; 1:1,000) antibody was purchased from Millipore. EZH2 phospho–Thr487 (EPR1410, ab109398; 1:1,000) was purchased from Abcam.