Extended Data Fig. 3: Co-immunoprecipitation and photo-affinity displacement assay with dipeptide degraders and ligands.
From: The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron
(a) Co-immunoprecipitation of endogenous BRD4 from HEK-CRBN in cells (left) or in lysates (right) after 2 h treatment with 25 µM or 1 µM of the indicated degrader. Western blot data are representative of 2 independent replicates. (b) Photo-affinity labeling displacement assay using photo-lenalidomide (pLEN) to visualize in-gel fluorescence imaging of CRBN/DDB1. Comparisons were performed using unpaired two-tailed t-tests. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Data are presented as mean ± SD (n = 3 biologically independent samples). (c) Structure of photo-lenalidomide (pLEN) and schematic of photo-affinity labeling displacement assay using photo-lenalidomide (pLEN) to visualize in-gel fluorescence imaging of CRBN/DDB1. (d) Corresponding gel images after treatment of CRBN/DDB1 with 1 µM pLEN and 100 µM of the indicated competitor for 30 min prior to photo-affinity labeling and in-gel fluorescence imaging. Data shown include 3 biologically independent replicates. For uncropped western blot and gel images, see Supplementary Fig. 9.
