Vector and 1 µg/mL NanoLuc-CRBN fusion vector, or 9 µg/mL Transfection Carrier DNA and 1 ug/mL VHL-NanoLuc fusion vector. Transfection complexes were allowed to form for 20 minutes at room temperature, then were added to 20 mL of HEK293 cells at density of 200,000 cells/mL. Transfected HEK293 cells were plated in a T75 flask and allowed to express protein overnight. The next day, 85 µL of transfected cells (at 200,000 cells/mL) were replated in white non-binding 96-well plates for live and lytic target engagement measurements. For live mode, a 100x solution of NanoBRET tracer was prepared using 100% DMSO (50 µM for CRBN live-cell, 100 µM for VHL live-cell). 100x tracer was used to prepare 20× tracer using tracer dilution buffer. PROTACs or test compounds were prepared at 10x final concentrations in Opti-MEM. 5 µL of the prepared tracer was then dispensed to each well and the plate was mixed on an orbital shaker at 300 RPM for 15 seconds. 10 µL of each 10x PROTAC dilution was added to the cells and the plate was mixed again before being incubated at a 37°C, 5% CO2 incubator for 2 or 5h (as indicated in each figure).