AlphaLISA assays. Ligands were titrated against 4nM His-tagged BRD4 BD2 and 10nM biotinylated JQ1. All reagents were diluted in 50mM HEPES, 100mM NaCl, 0.1% BSA, 0.02% CHAPS, pH7.5 (final concentration). On VCB premixed condition, the buffer also included 12.5µM VCB. Ligands were tested over an 11-point 3-fold serial dilution in duplicates for each concentration point, starting at 100µM without VCB or starting at 10µM with 20µM VCB, and giving a final DMSO concentration of 1%. Binding was detected using anti-His6 antibody-conjugated AlphaLISA acceptor beads and streptavidincoated donor beads (PerkinElmer), with a final concentration of 10µg/ml for each bead). Titrations were prepared in white 384-well Alphaplates (PerkinElmer), and read on a Pherastar FS plate reader (BMG) equipped with an AlphaLISA excitation/emission module. Data was analyzed and dose-response curves generated using GraphPad Prism. Each assay well had a final volume of 25µl. First 10µl of 2.5X ligand or 2.5X ligand with VCB was mixed with 5µl of a 5X mix of bromodomain and biotinylated JQ1 and incubated for 1h at room temperature. The assay plate was then moved to a dark room and 5µl of 5X acceptor beads were added and incubated for 1h. Then (still in darkness) 5µl of 5X donor beads were added, the plate was incubated for 1h before being read.