We next asked if degradation with 3 after irradiation could be observed within the same timeframe as the parent PROTAC 2, given that the caged molecule 3 requires an activation step before it can act as a degrader. After a 2 h-preincubation in HeLa cells, degradation of BRD4 was monitored for the first few hours following irradiation (Figure ). Interestingly, while PROTAC 4 took about 4h to fully knockdown BRD4, complete loss of the protein was seen just after 1 h at 1 μM for both 2 and 3. These results demonstrate that our caging strategy allows degradation to proceed within the same timeframe as for parent uncaged molecule 2 and rule out uncaging as a rate-limiting step. We next examined the mode of action of the caged PROTAC, which should function in an E3 ligasedependent and proteasome-dependent manner only after irradiation. To verify this hypothesis, HeLa cells were pre-incubated with increasing concentrations of VHL ligand 1 (0.1 to 10 μM) before being treated with caged or uncaged PROTACs. Saturation of VHL E3 ligase hampered BRD4 degradation by PROTACs 2 and 4 (Figure ), whilst the same inhibition of BRD4 degradation was observed for 3 with rising concentrations of ligand 1, but only following irradiation. In the same manner, proteasome inhibitor bortezomib (BTZ) prevented BRD4 degradation when the active degraders 2 and 4 were assessed irrespective of the presence or absence of UV light (Figure ). On the other hand, inhibition of the proteasome machinery affected the ability of 3 to knockdown BRD4 only following irradiation. These results confirm that our caging strategy in combination with irradiation does not affect the mode of action of the released degrader.