With our initial heterotrivalent PROTACs 1 and 2 in hand, we moved to evaluate the BET degradation profiles in cells. To this end, we performed live cell kinetic degradation assays in previously established CRISPR-edited HEK293 cell lines in which the 11-amino acid peptide, HiBiT, is appended to the N-terminus of endogenous BRD2, BRD3, and BRD4, and which stably express the 18 kDa LgBiT protein to produce NanoBiT luminescence. 23 We treated HiBiT-tagged BRD2, BRD3 and BRD4 HEK293 cells with varying concentrations of 1 and 2 (Figure ). Both 1 and 2 induced degradation of BRD2, BRD3 and BRD4, with Dmax 50s of 84 and 156 nM, respectively for BRD2; 23 and 21 nM, respectively for BRD3; and 37 and 55 nM respectively for BRD4. To assess whether each E3 ligase ligand of 1 and 2 was able to drive degradation, we ran a similar experiment by treating HiBiT-BRD4 HEK293 cells with 1 or 2, with or without pre-treatment of either CRBN ligand pomalidomide, 24 or VHL inhibitor VH298 (Figure ). 25 When VHL binding is blocked by VH298, the degradation profiles of 1 and 2 are not drastically affected by the inability to recruit VHL when comparing to the vehicle control, with Dmax 50s of 34 and 58 nM, vs 32 and 38 nM, respectively (Figure ). In contrast, when CRBN recruitment was outcompeted by pomalidomide binding, the degradation potency dropped by 5.3-fold in the case of 1 (Dmax 50s of 32 vs 166 nM), and by 3.6-fold in the case of 2 (Dmax 50s . This data suggests that although both 1 and 2 can degrade BRD4 in the absence of either VHL or CRBN, there is a strong preference for CRBN mediated degradation over VHL. We further assessed the contributions of each ligase warhead of 1 and 2 by monitoring the compound's antiproliferative effect in the BET sensitive, poorly differentiated colon carcinoma cell line, RKO, for which we had generated both CRBN and VHL single knockouts (KO), and CRBN/VHL double knockouts (dKO) (Figure & Supplementary Table ).