Histone extraction and Western blotting.

MEFs were lysed with the NIB-250 buffer (15 mM Tris-HCl [pH 7.5], 60 mM KCl, 15 mM NaCl, 5 mM MgCl₂, 1 mM CaCl₂, 250 mM sucrose) plus 0.3% NP-40 and inhibitors (PMSF, dithiothreitol [DTT], sodium butyrate, and sodium orthovanadate) at a ratio of 10:1. The nuclei were then rinsed with NIB-250 without NP-40, followed by acid extraction with 0.4 N sulfuric acid for longer than 3 h at 4°C. The histone-containing supernatant was then TCA precipitated on ice for 1 h, washed with acetone, and then air dried prior to solubilization with water and Western blot analysis. The histone samples were run on an SDS-PAGE gel (Invitrogen) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer.