Black 384 well low volume plates

All HTRF assays were performed by combining recombinant protein and synthetic histone peptide in assay buffer (25 mM HEPES pH 7, 20 mM NaCl, 0.2% Pluronic F-127, and 0.05% BSA) with 1 nM LanthaScreen Eu-anti-His Tag antibody (ThermoFisher, Cat. No, PV5597) and 8.9 nM SureLight allophycocyanin-streptavidin (Perkin Elmer, APC-SA, Cat. No. CR130-100). ENL YEATS, AF9 YEATS, and YEATS4 were used at 5 nM and BRD4 BD1 was used at 10 nM. ENL and AF9 assays were performed with H3(13-32)K27cr (13.3 and 100 nM, respectively), custom synthesized at ABclonal (N-terminal biotin, C-terminal amide); YEATS4 assay was performed with 25 nM H3(21-43)K27ac from Anaspec (Cat. No. AS-64846-1); and BRD4 BD1 assay was performed with 13.3 nM tetra-acetylated H4 (BioVision, Cat. No. 7144-01). Once all reagents were combined (with or without peptide), 10 μL was dispensed per well into black 384-well low-volume plates (Corning, Cat. No. 3821) and drug was added by pin tool transfer (Biomek FX). Assays were incubated for 2 or more hours before measurement of HTRF signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual emission; excitation = 337 nm, emission 1 = 665 nm, emission 2 = 620 nm). HTRF signal (ratio of emission 1 to emission 2) from vehicle-treated wells (maximum signal) and no-peptide-control wells (minimum signal) were used to calculate percent inhibition for drug-treated wells.