Purification and HAT Assay of Protein Complexes

Purification of protein complexes after co-transfection was performed as previously described (26 (link),27 (link)). Briefly, 293T cells were transfected with each subunit (HA-BRPF1 (wt or mutants), Flag-MOZ, Flag- or HA-ING5 and Flag- or HA-hEaf6) and harvested 48 h later. Flag or HA IP using anti-Flag M2 agarose beads (Sigma) or anti-HA agarose beads (Roche) was performed followed by elution with 3× Flag or HA peptides. HAT assays were performed as described previously (22 (link)). Briefly, native human chromatin and free histones were used to carry out HAT assays in a 15 μl reaction containing 50 mM Tris–HCl pH 8.0, 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, 10 mM sodium butyrate (Sigma) and 1.25 nCi ³H labeled acetyl-CoA (Perkin Elmer Life Sciences). Samples were spotted on P81 membranes (GE Healthcare) for scintillation counting.