In vitro methylation assay

Methylation assay reactions contained 4 μg of His or His-Sumo BRD4 1-477aa wild type, BRD4 K99R mutant, 1 μg of His BD1 or BD2, and 4 μg of His SETD6 or GST SETD6, 2 mCi of ³H-labeled SAM (Perkin-Elmer, AdoMet), and PKMT buffer [20 mM tris-HCl (pH 8), 10% glycerol, 20 mM KCl, and 5 mM MgCl₂]. The reaction tubes were incubated overnight at 30°C. The reactions were resolved by SDS-PAGE for Coomassie staining (Expedeon, InstantBlue) or autoradiography. For the nonradioactive (cold) methylation assay, the ³H-labeled SAM was switched to 300 μM cold SAM (Abcam, ab142221).