Ab50818

HeLa cells were seeded on coverslips at low density in Opti-MEM medium and then transfected with the appropriate siRNA using Lipofectamine RNAiMAX as per the manufacturer protocol, see STAR Methods and Table S1C for details. 48 hr later, cells were fixed with 3.7% paraformaldehyde/PBS and permeabilized in 0.3% Triton X-100 in PBS. Mouse anti-BRD3 antibody (1:100; ab50818, Abcam) and anti-TCOF1 (1:500; HPA038237; Sigma-Aldrich) were used to identify BRD3 and TCOF1, respectively. Proteins were visualized with goat anti-mouse or anti-rabbit coupled to Alexa Fluor 488 or 555 antibodies (1:1,000; A11001">A11001, A11008">A11008, A21422">A21422, A21428">A21428; Invitrogen). DNA was detected with DAPI staining. Immunofluorescence was observed by confocal microscopy on a Nikon Eclipse C1si instrument.

Cells were seeded (HEK293: 1
× 10⁵ cells/well) in 12-well plates. Following compound
treatment, cells were lysed on ice with RIPA lysis and extraction
buffer (Thermo Fisher Scientific, 89901) supplemented with protease
inhibitor cocktail (Merck, 11697498001) and Benzonase Nuclease (Sigma-Aldrich,
E1014). Protein concentration was determined using the BCA assay (Thermo
Fisher Scientific, 23225). Samples were then prepared and loaded onto
NuPAGE 4–12% Bis–Tris Midi gels (Thermo Fisher Scientific,
WG1403A) followed by the transfer of the proteins onto nitrocellulose
membranes (EMD Millipore). The membranes were blocked for 1 h prior
to incubation with the primary antibodies using 5% Milk TBST. Membranes
were probed for Brd2 (Abcam, Ab139690, 1:1000), Brd3 (Abcam, Ab50818,
1:4000), and Brd4 (Abcam, Ab128874, 1:1000). Following overnight incubation
with the primary antibodies at 4 °C, the membranes were incubated
with secondary antibodies (Anti-rabbit, Abcam AB216773, 1:5000 or
antimouse, Abcam AB216774, 1:5000) and hFAB Rhodamine Anti-Tubulin
Antibody (Bio-Rad, 12004165, 1:10 000) for 1 h and then imaged
with a Bio-Rad imager (LI-COR Biosciences). All Western blots were
analyzed for band intensities using Image Lab from Bio-Rad (LI-COR
Biosciences). The data extracted from these blots were then subsequently
plotted and analyzed using Prism (v. 8.2.1, GraphPad).