NanoBRET Ubiquitination, Ternary Complex, and Biosensor Experiments. For endogenous live cell BET:Ubiquitin,BET:VHL, and BET:CRBN assays, CRISPR HiBiT-BRD2, HiBiT-BRD3, and HiBiT-BRD4 HEK293 cells stably expressing LgBiT were transfected with 2µg of HaloTag-UBB,HaloTag-VHL, or HaloTag-CRBN vectors in 6-well plates using FuGENE HD (Promega). For full transient NanoBRET experiments with NanoLuc-BRD4 WT, N433F, or N140F mutants, HEK293 cells (8 ×10 5 ) were co-transfected with 0.02µg NanoLuc-BRD4 and 2µg of HaloTag-VHL vectors. For transient NanoBRET experiments with the BRD4 NL-BD1-BD2-HT biosensor containing either the WT tandem BD1-BD2 domain the N433F mutation, HEK293 cells (8 ×10 5 ) were transfected with 0.02µg biosensor plasmid and 2µg carrier DNA. The following day, transfected cells (2 × 10 4 ) were replated in quadruplicate into white 96-well tissue culture plates in the presence or absence of HaloTag NanoBRET 618 Ligand (Promega) and incubated overnight at 37°C, 5% CO2. For kinetic experiments, medium was replaced with Opti-MEM (Gibco) containing a 1:100 dilution of Vivazine (Promega), and plates were incubated at 37 °C, 5% CO2, for 1h before addition of DMSO or 10nM-1µM final concentration of the indicated compounds. Continual BRET measurements were then made every 3 min up to 5h on a CLARIOstar equipped with an atmospheric control unit (BMG Labtech) set to 37 °C and 5% CO2. For the biosensor experiments, the cells were treated with a 3-fold serial titration of 10µM of the indicated compounds. NanoBRET NanoGlo (Promega) substrate was added and BRET was measured two hours post-compound treatment using a GloMax Discover Microplate Reader (Promega). Dual filtered luminescence was collected with a 460/80nm bandpass filter and a 610nm long pass filter (acceptor, HaloTag NanoBRET ligand) using an integration time of 0.5 s. For all NanoBRET experiments, background subtracted NanoBRET ratios expressed in milliBRET units.Fold increase in BRET was calculated by normalizing mBRET ratios to the average mBRET ratios for DMSO controls.