Next, we wanted to compare how intracellular ubiquitination levels in parental, CRBN KO, and VHL KO HiBiT-BRD4 cells differed (Figure ). To this end, we adopted a NanoBRET ubiquitination assay similar to the ternary complex assay described previously. In the NanoBRET ubiquitination assay, parental, CRBN KO, or VHL KO HiBiT-BRD4 cell lines were transiently transfected with HaloTag-Ubiquitin and treated with a dilution series of 27. 11, 40 Ubiquitination of BRD4 in parental cells treated with 27 occurs more rapidly than in either the CRBN KO or VHL KO cells, and the parental cells also exhibit a larger magnitude in BRET fold-change. Taken together, this indicates that each ligase is contributing to ubiquitination and therefore helping to drive the degradation of BRD4 when cells are treated with 27.