I-BET151 (GSK1210151A) (GSK1210151A) is a specific BET inhibitor for BRD2/3/4 (IC50: 0.5/0.25/0.79 μM, in cell-free assays).

BRD4:0.79 μM, BRD2:0.5 μM, BRD3:0.25 μM

Fluorescence anisotropy (FP) ligand displacement assay: All components are dissolved in buffer of composition 50 mM HEPES pH 7.4, 150 mM NaCl and 0.5 mM CHAPS with final concentrations of BRD 2/3/4 75 nM, fluorescent ligand 5 nM. 10 μL of this reaction mixture is added using a micro multidrop to wells containing 100 nL of various concentrations of I-BET151 or DMSO vehicle (1% final) in Greiner 384 well Black low volume microtitre plate and equilibrated in the dark for 60 minutes at room temperature. Fluorescence anisotropy is read in Envision (lex = 485 nm, lEM = 530 nm; Dichroic = 505 nM).

Cells are exposed to various concentrations of I-BET151 for 24 or 72 hours in 384-well or 96-well plates. For cell growth inhibition assays, plates are added with CellTiter-Glo reagent using a volume equivalent to the cell culture volume in the wells, shaken for approximately 2 minutes and chemiluminescent signal is read on the Analyst GT or EnVision Plate Reader. For cell proliferation assays, CellTiter-Aqueous One is added to each well and plates are incubated for 4 hours at 37 °C. Absorbance is read at 490 nm on a SpectraMax Gemini reader (Only for Reference)

GSK1210151A

DMSO: 55.5 mg/mL (133.59 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.81 mM), Sonication is recommended.