Figure 5
From: Disordered region of cereblon is required for efficient degradation by proteolysis-targeting chimera
The N-terminal disordered region of CRBN is necessary for degradation, but not for ubiquitination, by VHL-CRBN heterodimerizing PROTACs. (A) Schematic diagram illustrating CRBN truncation mutants. LON, Lon protease domain; TB, thalidomide binding domain. (B) Xpress-tagged full-length CRBN or D1, D2, D3, or D4 deletion mutants were expressed in HEK293T cells. After 24 h, the cells were treated with TD-165 (3 μM) or DMSO for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins. (C) Plasmids encoding Xpress-tagged D1 and His-SBP–tagged VHL were transfected into HEK293T cells. After 48 h, cells were treated with TD-165 (1 μM) or DMSO for 24 h. Whole-cell lysates and proteins pull-downed using streptavidin beads were analyzed by immunoblotting for the indicated proteins. (D) Plasmids encoding Xpress-tagged CRBN, K39R, K42/43 R, or K39/42/43 R mutants were transfected into HEK293T cells. After 24 h, the cells were treated with TD-158 (2 μM) or DMSO for 24 h. Whole-cell lysates were analyzed by immunoblotting for the indicated proteins.
