Ab128874

RH4-BRD4-mGFP were seeded at 85,000 per well into chambered cover glass (ThermoFisher #155379PK, Waltham, MA, USA). The following day, they were treated with 100 nM ARV-771 or an equal volume of DMSO (0.1%). Then, 6 hours later, they were fixed with 1 mL of 4% formaldehyde for 14 min at RT, and quenched with 200 µL of glycine for 5 min at RT. They were subsequently washed with PBS 3x, permeabilized with 0.5% Triton X-100 in TE pH 7.4 for 15 min at RT, washed with PBS 3x, and blocked with 4% FBS in PBS-T (PBS + 0.1% Triton X-100) for 1 h at RT. The following primary antibodies and dilutions were then applied and incubated overnight at 4 °C: rabbit anti-BRD4 (Abcam #ab128874, Cambridge, UK) 1:200, mouse anti-MyoD (Invitrogen #MA1-41017, Waltham, MA, USA) 1:200 in PBS-T. The next day, slides were washed 3x with PBS-T, incubated for 1 h at RT protected from light with secondary antibodies, and washed again. Secondaries included anti-rabbit 594 (Cell Signaling #8889S, Boston, MA, USA) and anti-mouse 488 (ThermoFisher #A-21121) diluted 1:1000 in PBS-T. Slides were then stored in PBS at 4 °C until imaging on a Leica TCS SP8 gated STED microscope.

Total fraction proteins were separated using 10% SDS-PAGE with Tris-Gly buffer and transferred to 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 1 h at 250 mA on ice. The assessment of the quality of protein transfer, hybridization with primary and secondary antibodies, and protein development were performed as described previously. For primary hybridization, rabbit antibodies (Abcam, Cambridge, UK) to SUV39H1 (ab245380, 1:3000), SUV39H2 (ab229493, 1:3000), G9a (ab183889, 1:3000), DOT1L (ab64077, 1:500), EZH2 (ab228697, 1:7000), BRD2 (ab139690, 1:10,000), BRD3 (ab264294, 1:5000), BRD4 (ab128874, 1:1000), and DNMT3a (ab2850, 1:500); rabbit antibodies (Invitrogen, Carlsbad, CA, USA) SUV420H1 (PA5-40926, 1:3000) and SUV420H2 (PA5-109891, 1:3000); and rabbit antibodies DNMT1 (ABclonal, San Diego, CA, USA, A5495, 1:3000) were used. Goat antibodies (Abcam, ab97051, 1:10,000) were used for secondary hybridization. To control protein loading, rabbit antibodies to β-actin (Abcam, Cambridge, UK, ab227387, 1:5000) were used. All experiments were performed at least three times.