Cell culture, cell treatments and western blotting for BRD4 degradation

HEK293 cells were obtained from Genentech’s repository and were cultured under standard conditions in RPMI medium containing 10% FBS. For evaluating the cellular effects of the degrader compounds, 0.4 million cells were seeded in 1 well of a 12-well plate. The following day, the medium was diluted twofold with fresh medium containing vehicle (DMSO) or compounds. For degradation rescue studies, cells were co-treated with the proteasome inhibitor bortezomib (62.5 nM) or BET ligand (0.1, 1 or 10 µM). Cells were cultured with the compounds for an additional 20 h and collected. Complete cell lysates were prepared in urea lysis buffer (50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 1 mM EDTA, 1 % NP-40, 6 M urea and 1× Roche cOmplete protease inhibitor cocktail). Ten micrograms of total lysate was resolved by NuPAGE 3–8% Tris-acetate gels (Thermo Fisher) and transferred to nitrocellulose membranes (Bio-Rad Trans-Blot transfer system) followed by western blotting analysis. Primary antibodies were diluted 1:3,000 and incubated for 1 h at room temperature or 4 °C overnight. Secondary antibodies were diluted 1:10,000 and incubated for 60 min at room temperature. Blots were imaged by scanning using LICOR Odyssey CLx.