Extended Data Fig. 9: Effects of tryptoline acrylamide ligands on protein interactions of N112C-CCNE1:CDK2 complexes.
From: An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes
a, Western blotting of enriched CKS2 in the indicated anti-Flag IP experiments performed as described in Fig. 5d. Top, bar graph depicting quantification of CKS2 enrichment blots and represent average values ± s.e.m. from three biological replicates. Unpaired, two-sided t test with Welch’s correction was perform to test statistical significance. b, Scheme of in cellulo NanoBRET assay measuring CCNE1:CKS1B and CCNE1:CKS2 interactions. c, NanoBRET data showing stabilization of Flag-N112C-CCNE1:CKS1B and:CKS2 interactions, but not Flag-WT-CCNE1:CKS1B and:CKS2 interactions by WX-02-308 (20 µM 6 h). Also shown are NanoBRET data for three representative nuclear proteins (PCBP1, DDX21, RBBP7) not known to interact with CCNE1:CDK2 complexes as negative controls. Data represent average values ± s.e.m. from four independent replicates. Unpaired, two-sided t test with Welch’s correction was used to test statistical significance. d, NanoBRET data showing WX-02-308, but not WX-02-326 or I-125A, stabilizes Flag-N112C-CCNE1:CKS1B and:CKS2 interactions in a concentration-dependent manner. Data represent average values ± s.e.m. from four biological replicates. e, Overlay of crystal structures of CKS1B:CDK2 (PDB: 1BUH) and CCNE1:CDK2 (PDB: 5L2W) showing the relative locations of CCNE1_N112 and CKS1B binding site on CCNE1.
