NanoBRET Target Engagement and Residence Time. For target engagement experiments in live and permeabilized cells, CRISPR HiBiT-BRD2 and BRD4 HEK293 cells stably expressing LgBiT or HEK293 cells transfected with VHL-NanoLuc fusion (Promega N275A) were plated into white 96-well tissue culture plates at a density of 2 × 10 4 cells/well. Cells were equilibrated for 1h for BET family experiments and 5h for VHL with energy transfer probes and the indicated test compound prior to NanoBRET measurements. NanoBRET tracers were prepared at a working concentration of 20X in tracer dilution buffer (12.5mM HEPES, 31.25% PEG-400, pH 7.5). To measure NanoBRET in live cells, NanoBRET BRD Tracer-02 was added to cells at a final concentration of 0.5µM for BET family experiments and for VHL target engagement assays, NanoBRET VHL tracer was added at a final concentration of 1µM. NanoBRET NanoGlo Substrate and Extracellular NanoLuc Inhibitor (Promega) for VHL assay were added according to the manufacturer's recommended protocol, and filtered luminescence was measured on a GloMax Discover Microplate Reader (Promega) equipped with 450 nm BP filter (donor) and 600 nm LP filter (acceptor) using 0.3 s integration time. To measure NanoBRET in permeabilized cells, digitonin was added to the cells to a final concentration of 50µg/mL, tracer was added to a final concentration of 0.25µM, and Extracellular NanoLuc inhibitor was omitted during the detection step. Tracer and test compounds were incubated for 10 min prior to measuring BRET. For residence time experiments CRISPR HiBiT-BRD2 and BRD4 HEK293 cells stably expressing LgBiT were trypsinized, washed and resuspended to a density of 2 × 10 5 cells/ml in Opti-MEM and incubated with either 1µM JQ1, 1µM SIM1, 100nM cis-SIM1, 10µM (R,S)-SIM1, 100nM MT1, 10µM MZ1 or 10µM cis-MZ1 representing the representative IC80 values for tracer displacement in live cell format. Cells were incubated in 15mL conical tubes with caps loosened in a tissue culture incubator for 1h. Following incubation, cells were spun at 300x g for 5 min, washed once with Opti-MEM, spun a second time at 300x g for 5 min, then resuspended with fresh Opti-MEM before plating at 2 × 10 4 cells/well. NanoBRET BRD Tracer-02 was added at a final concentration of 0.5µM cells and kinetic NanoBRET measurements were collected. NanoBRET ratios were expressed in milliBRET units and calculated according to the equation in the NanoBRET Ubiquitination, Ternary Complex and Biosensor Experiments section.