AF9 YEATS, BRD4 BD1, and ENL YEATS HTRF assays
were performed exactly as previously described.33 (link) All assays were conducted by combining recombinant AF9
YEATS (5 nM), BRD4 BD1 (10 nM), or ENL YEATS (5 nM) with synthetic
histone peptide (100 nM H313–32K27cr custom synthesized
by ABclonal, 13.3 nM tetra-acetylated H4 from BioVision Cat. No. 7144–01,
or 13.1 nM H313–32K27cr, respectively), 1 nM LanthaScreen
Eu-anti-His Tag antibody (ThermoFisher, PV5597), and 8.9 nM SureLight
allophycocyanin-streptavidin (PerkinElmer, CR130–100). For
the primary screen, the assay mixture was dispensed into black 1536-well
plates (Greiner HiBase) at 6 μL/well and compounds were added
by acoustic transfer (Labcyte, Echo Liquid Handler). Dose–response
assays were performed with 10 or 20 μL assay volumes in black
384-well low-volume plates (Corning, Cat. No. 3821), and compounds
were added by pin tool transfer (Biomek FX). Compounds were incubated
with the assay mixture for at least 2 h before measuring the HTRF
signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual
emission; excitation = 337 nm; emission 1 = 665 nm; emission 2 = 620
nm). The HTRF signal was calculated as the ratio of emission 1 to
emission 2. Each plate included samples treated with vehicle (DMSO)
or lacking the peptide substrate, which were used to calculate the
percent inhibition for compound treated wells.
were performed exactly as previously described.33 (link) All assays were conducted by combining recombinant AF9
YEATS (5 nM), BRD4 BD1 (10 nM), or ENL YEATS (5 nM) with synthetic
histone peptide (100 nM H313–32K27cr custom synthesized
by ABclonal, 13.3 nM tetra-acetylated H4 from BioVision Cat. No. 7144–01,
or 13.1 nM H313–32K27cr, respectively), 1 nM LanthaScreen
Eu-anti-His Tag antibody (ThermoFisher, PV5597), and 8.9 nM SureLight
allophycocyanin-streptavidin (PerkinElmer, CR130–100). For
the primary screen, the assay mixture was dispensed into black 1536-well
plates (Greiner HiBase) at 6 μL/well and compounds were added
by acoustic transfer (Labcyte, Echo Liquid Handler). Dose–response
assays were performed with 10 or 20 μL assay volumes in black
384-well low-volume plates (Corning, Cat. No. 3821), and compounds
were added by pin tool transfer (Biomek FX). Compounds were incubated
with the assay mixture for at least 2 h before measuring the HTRF
signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual
emission; excitation = 337 nm; emission 1 = 665 nm; emission 2 = 620
nm). The HTRF signal was calculated as the ratio of emission 1 to
emission 2. Each plate included samples treated with vehicle (DMSO)
or lacking the peptide substrate, which were used to calculate the
percent inhibition for compound treated wells.
