To further evaluate the observed hook effect seen in the cell viability assay, we decided to orthogonally investigate this in cell degradation assays by western blot, assessing Brd4 protein levels in HEK293 cells starting with a 10 µM treatment of PROTAC (Figure ). Strikingly, ester compounds 26 and 27, and also amide 22, exhibited a hook effect at 10 µM. Compounds 22 and 26 both possess an extra methyl on the VH032 ligand, which enhances binary binding affinity to VHL. A possible explanation for the observed onset of the hook effect in 22 and 26 is that their stronger binding to VHL contributes to the binary complex being more effective at outcompeting the ternary complex formation. Alternatively, this could also be attributed to the increased lipophilicity and permeability conferred by the added methyl group (cf. 22 vs 24 and 26 vs 28, Table , SI Table ). Similarly, 26 appears to hook to a greater extent than its amide counterpart 22, suggesting that intracellular concentrations of 26 are higher, most likely due to the increase in lipophilicity and PAMPA permeability (cf. Table , Figure , SI Table ). Interestingly, 27 is the only non-methylated VH032-based compound that exhibited the hook effect. This could be due to 27 being the most lipophilic compound out of the series (ALogP = 5.5).