Fig. 3
Cellular AlphaLISA assay development to detect bromodomain–histone H3 interactions in HTS format. a Schematic diagram for quantifying the association of TRIM24 and histone H3 in cells using AlphaLISA. Streptavidin-coated donor beads capture the immune complex between the biotinylated anti-H3 antibody and histone H3. Acceptor beads (directly conjugated to either anti-FLAG or anti-V5 antibodies) captures TRIM24. If a test agent displaces TRIM24 from histone H3, it will lead to decreased Alpha Signal. b, c Stringency of histone extraction evaluated for HeLa TRIM24-PB cells pretreated with either DMSO or SAHA (5 µM, 2 h) using the indicated AlphaLISA acceptor beads (V5 or FLAG) and a dilution series of the histone extraction buffer. d Titration of the anti-histone H3 antibody with FLAG-acceptor beads (5 mg/mL). e SAHA time-course (5 µM) and f dose–response (2 h) studies for TRIM24-PB HeLa cells plated in 384-well plates and subjected to the AlphaLISA protocol (Additional file 1: Table S2). g Dose–response curves for cells treated with IACS-6558, the depicted small-molecule TRIM24 bromodomain inhibitor (TRIM24i). Inhibitor affinity (EC50) is shown as percent binding relative to control (DMSO) from 6 independent experiments. h Correlation plot between biochemical AlphaScreen (IC50) and cellular AlphaLISA (EC50) values for 273 compounds evaluated during our TRIM24 bromodomain drug discovery program [14]