To profile the degradation activity of our panel of PROTACs, VHL and CBRN protein levels in HeLa cells were quantified by Western Blot analysis following a 4 h treatment with 1 µM compounds, using CM11 and CC15a as positive controls for VHL and CRBN degradation, respectively (Figure ). Interestingly, we observed significant degradation of CRBN with a few compounds, while no significant degradation of VHL was observed with any of the compounds tested. The most profound CRBN degradation was observed with PROTAC 14a (64% protein degradation, as quantified by western blot), followed by compound 18b which induced CRBN degradation to a lower extent (54% degradation). The same screen was conducted in a different cell line (HEK293), confirming 14a as the most potent compound at inducing CRBN degradation (data not shown). To provide a more stringent screen the same experiment was conducted by testing compounds at 10 nM in HeLa cells (Figure ). The purpose of this experiment was to exclude the possibility of dismissing any potent compound as a false negative potentially due to the "hook-effect" characteristic of bivalent molecules: whereby unproductive binary complexes preferentially form at high PROTAC concentration, which compete with and eventually suppress the formation of a productive ternary complex. PROTAC 14a induced less CRBN degradation (19% protein degradation) at 10 nM compared to 1 µM, as expected. However, CRBN protein levels remaining at 10 nM were not significantly lower than the levels remaining after the same treatment at 1 µM (cf. Figure with Figure ), making it unlikely that any of the compounds might be false negatives due to a hook effect. Interestingly, at this lower concentration some compounds appeared to induce up to 50% degradation of pVHL30 (Figure ). This suggests that depending on the concentration being used this class of compounds could preferentially induced the depletion of one ligase over the other. Encouraged by the promising and consistent degradation of CRBN observed with PROTAC 14a, we selected this compound for further characterization. We next profiled the concentration-and time-dependent activity of 14a in both HeLa (Figure ) and HEK293 cells (Figure ). Compound 14a degraded CRBN with a half-degrading concentration DC 50 (i.e. the concentration causing 50% reduction of protein level relative to vehicle) of 200 nM, and reached a maximal degradation (D max ) of 75% after 4 h treatment with 1 µM. A "hook-effect" was observed above 1 µM, indicating that 14a preferentially forms the 1:1 species (i.e. acts as inhibitor) at higher concentrations. Very similar degradation profile and comparable DC 50 and D max were found for 14a in HEK293 (Figure ). Again, some concentration-dependent depletion of pVHL30 was seen at the lower end of the concentration range (5-50 nM) in HeLa (Figure ). However interestingly this effect was not observed in HEK293 (Figure ).