Rbbp4

One hundred micrograms of lyophilized histone peptides (H3K4ac, Epicypher; unmodified H3, H3K4me1, H3K4me2, H3K4me3; Millipore) were dissolved in 400 µL of PBS. 1 × 10⁸ MEFs were hypotonically lysed with Buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, freshly added protease inhibitors and DTT to 1 mM) before nuclear extract was isolated with Buffer C (20 mM HEPES at pH 7.9, 25% v/v glycerol, 420 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, freshly added protease inhibitors and DTT to 1 mM) and the salt concentration lowered to 150 mM using Buffer D (20 mM HEPES at pH 7.9, 20% v/v glycerol, 0.2 mM EDTA, 0.2% Triton X-100). Following mechanical lysis by sonication, 50 µL of prewashed avidin-histone peptide slurry was added to precleared lysate and incubated overnight at 4°C. After washing of the avidin beads, SDS sample buffer was added and samples were boiled for 5 min and subsequently run on a 10% SDS gel. The following antibodies were used for the histone peptide pull-down assay: mSin3A (Abcam, ab3479), CoREST (Millipore, 07–455), HDAC2 (Abcam, ab7029), HDAC1 (Abcam, ab7028), RBBP4 (Abcam, ab79416), MTA1 (Santa Cruz, sc-9446).