Streptavidin d2

The biotinylated tetra-acetylated histone H4 peptide (H4Ac₄, N-terminal H4 peptide, residues 1–20, with acetylation of K5, K8, K12 and K16) was purchased from Covalab (Villeurbanne, France). HTRF reagents and buffers were purchased from Cisbio. HTRF assays were performed as previously described. Mietton, 2017 #16} Briefly, GST-tagged bromodomains (human Brd4 BD1 and BD2, CaBdf1 BD1 and BD2) in 25 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM DTT were assayed at 5 nM final concentration. The corresponding final concentrations of biotinylated H4Ac₄ peptide used were 50, 600, 300 and 400 nM, respectively. The antibody-conjugated donor (Mab anti-GST-Tb; Cisbio) was used at 0.5 nM and the streptavidin-conjugated acceptor (streptavidin-d2; Cisbio) was used at 1/8 of the H4ac₄ peptide concentration. Inhibitors were tested by performing a nine-point dilution series with a final concentration ranging between 0.013 and 20 μM). Experiments were performed in 384-well white plates (Greiner 781080) in a final volume of 16 μL. Plates were incubated at 4°C for 4 h (BD1) or 24 h (BD2) and subsequently analyzed in a ClarioStar plate reader (BMG LABTECH). Excitation was at 330 nm and emission intensities were measured at 620 and 665 nm with an integration delay of 60 μs and an integration time of 400 μs.