El406 liquid handler

Assays were performed with minor modifications from the manufacturer’s protocol (Perkin Elmer, USA). All reagents were diluted in AlphaScreen™ buffer (50 mM HEPES, 150 mM NaCl, 0.01% v/v Tween-20, 0.1% w/v BSA, pH = 7.4). After addition of Alpha beads to master solutions, all subsequent steps were performed under low light conditions. A 2x solution of components with final concentrations of His-Brd4(1) at 0.020 μM, Ni-coated Acceptor Bead at 10 μg/ml, and biotinylated-(+)-JQ1 at 0.010 μM were added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer) using an EL406 liquid handler (Biotek, USA). Plates were spun down at 1000 rpm. A 10-point 1: 3 serial dilution of compounds in DMSO was prepared at 200x the final concentration. 100 nL of compound from these stock plates were added by pin transfer using a Janus Workstation (PerkinElmer). A 2x solution of streptavidin-coated donor beads with a final concentration of 10 μg/ml was added in a 10 μL volume. The plates were spun down again at 1000 rpm and sealed with foil to prevent light exposure and evaporation. The plates were then incubated at room temperature for 1 h and read on an Envision 2104 (PerkinElmer) using the manufacturer’s protocol. IC50 values were calculated using a 4-parameter logistic curve in Prism 6 (GraphPad Software, USA) after normalization to DMSOtreated negative control wells (0.2% DMSO, v/v).

Fibrin-coated 384-well plates were prepared with a EL406 liquid handler (BioTek), columns 1-22 received 30 μL of 2 U/mL Thrombin (Sigma-Aldrich) in a buffer of 20 mM HEPES, pH 7.4, and 14 mM CaCl₂, followed by 30 μL of 12 μg/mL human plasminogen-free fibrinogen (EMD Milipore) in 20 mM HEPES, pH 7.4 for a final concentration of 6 μg/mL of fibrinogen. After incubation for 90 min in 37°C to allow fibrin formation, plates were dried overnight in a 37 °C incubator equipped with fans to circulate the air.