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. Author manuscript; available in PMC: 2016 Jun 18.
Published in final edited form as: Chem Biol. 2015 Jun 4;22(6):755–763. doi: 10.1016/j.chembiol.2015.05.009

Figure 2. Hijacking the E3 Ubiquitin Ligase Cereblon to create PROTAC to efficiently degrade BRD4.

Figure 2

(A) A schematic representation of bifunctional PROTAC ARV-825.

(B) ARV-825 leads to fast and efficient degradation of BRD4. (top panel) Namalwa and CA-46 cells were treated overnight with increasing doses of ARV-825, lysates were analyzed for BRD4 levels by immunoblot with actin serving as loading control; (bottom panel) Namalwa and Ramos cells were treated with ARV-825 (0.1 µM) for indicated time points, lysates were collected and subjected to immunoblot analysis with antibodies for BRD4 and actin.

(C) Confirmation of cereblon-based mechanism in driving BRD4 degradation upon ARV-825 treatment. Namalwa (left panel) and Ramos (right panel) cells were treated overnight with various concentrations of ARV-825 or pomalidomide (10 µM), or combination of ARV-825 and pomalidomide; lysates were analyzed by immunoblot for BRD4 and actin.

(D) Confirmation of proteasome-based mechanism in driving BRD4 degradation upon ARV-825 treatment. Namalwa cells were treated overnight with ARV-825 (+/0.01 µM and ++/0.1 µM) alone, MG132 (5 µM) or carfilzomib (5 µM) alone, or combination of ARV-825 with MG132 or with carfizomib; lysates were collected and analyzed by immunoblot for BRD4 and actin.