Extended Data Fig. 2 ∣. Evaluation of JQ1-XcQ and JQ1-XcN degraders in targeted protein degradation of BRD4.

(a) Western blots of BRD4 levels after treatment of HEK293T cells with 100 nM, 1 μM, or 10 μM dBET6 for the 20 dipeptide degraders. Interestingly, the hook effect resulting in reduced degradation of BRD4 can be observed from dBET6 at 10 μM, but not from the dipeptide degraders. (b) BRD4 degradation with dBET6 over 4 h was competitively inhibited by lenalidomide or Boc-FcQ over a dose-dependent concentration (0.1–100 μM), indicating that the thalidomide-binding domain of CRBN is engaged by glutarimide ligands. (c) Levels of BRD4 over time in HEK293T cells treated with dBET6 or JQ1-FcQ. (d) Evaluation of JQ1-HcN in targeted protein degradation of BRD4 at various concentrations in HEK293T cells over 4 h. This probe was constructed given that protein splicing products (e.g., inteins) are typically promoted by a penultimate histidine. (e) Western blot of BRD4 after treatment of wild type (WT) or shRNA knockdown (CRBN KD) HEK293T cells with the indicated degrader. (f) Western blot of BRD4 after co-treatment of HEK293T cells with JQ1-FcN and lenalidomide or Boc-FcN. (g) Levels of BRD4 over time in HEK293T cells treated with JQ1-FcN. All western blot data are representative of 2 independent replicates. For uncropped western blot images, see Supplementary Fig. 8.