Figure 2.

Degradation of CRBN by VHL-CRBN heterodimerizing PROTACs is dependent on CRL2^VHL. (A) HEK293T cells were treated with TD-165 (1 μM) for 12 h, followed by addition of bortezomib (20 nM) or DMSO for 8 h. Whole-cell lysates were subjected to immunoblot analysis for the indicated proteins. (B) FLAG-tagged CRBN (CRBN-Flag) and HA-tagged Ubiquitin (HA-Ub) were expressed in HEK293T cells. After 24 h, the cells were treated with TD-158 (500 nM) and bortezomib (20 nM), or DMSO and bortezomib (20 nM), for 12 h. Whole-cell lysates and proteins immunoprecipitated using Flag M2 magnetic beads were analyzed by immunoblotting for the indicated proteins. (C) HEK293T cells were transfected with siRNA specific for VHL or scrambled (control) siRNA. After 24 h, the cells were treated with TD-158 (500 nM) for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (D) 786-O cells and VHL-expressing 786-O cells (786-O + VHL) were treated with TD-158 (500 nM) for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (E) HEK293T and CUL2- knockdowned HEK293T cells were treated with TD-165 (1 μM) or TD-487 (1 μM) for 24 h and whole-cell lysates were analyzed by immunoblotting. (F) CRBN-Flag was expressed in HEK293T cells. After 36 h, the cells were treated with TD-158 (1 μM) and bortezomib (20 nM), or DMSO and bortezomib (20 nM), for 12 h. Whole-cell lysates and proteins immunoprecipitated using Flag M2 magnetic beads were analyzed by immunoblotting for the indicated proteins. (G) Purified CRBN and VHL/ELOB/ELOC complex proteins were mixed and aliquoted into four tubes. TD-158 and glutathione beads were added as indicated and incubated for 3 h. After incubation, glutathione beads were washed and analyzed by immunoblotting and Coomassie Blue staining.