Journal: bioRxiv

Article Title: The structural basis of Indisulam-mediated recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 ligase complex

doi: 10.1101/737510

Figure Lengend Snippet: a , Bioinformatics workflow for identifying proteins bearing putative degron motif required for DCAF15-Indisulam recruitment (left) and structures of DCAF15-bound Indisulam (orange) and RBM39(RRM2) (magenta) with central alpha helix highlighted (right). The RBM39 residues found to be most critical for DCAF15-Indisulam recruitment are labeled (M265, G268, E271, and P272). 6,475 proteins with known structures were identified in the Swiss-Prot database, of which 3,425 had a glycine in an alpha helix. 3,112 of the glycine-containing alpha helices aligned to an RBM39(RRM2) structure (2JRS) with RMSD <2.0 Å. Among these matches, only RBM23 and RBM39 helices had a sequence matching the required X 1 XXM 4 XXG 7 XXEP motif. RMSD values, PDB IDs, and gene names shown in bottom table. b , Volcano plot summary of expression proteomics experiments comparing lysates from HCT-116 cells treated for 4 h with 10 µM Indisulam or DMSO. Significant downregulated proteins (p value < 1E-2, Log 2 fold-change) are labeled. Data represents two (n=2) biological replicates per treatment condition in a single experiment. c , Western blots showing levels of RBM39, RBM23, and ZNF277 in HCT116 cells following 4 h treatment with varied concentrations of Indisulam or DMSO. Data shown from one individual, representative experiment from three independent repeats.

Article Snippet: Blots were incubated with primary antibody solutions made in TBS-T with 5% milk for RBM39 (Sigma, Cat #HPA001591, 1:2500) GAPDH (CST, Cat #2118L, 1:1000), Vinculin (Cell Signaling Technology, Cat# 13901S, 1:1000), ZNF277 (Pro-Sci, Cat #46-616, 1:1000), RBM23 (Invitrogen, Cat# PA5-52060, 1:1000), or DDA1 (Proteintech, Cat #14995-1-AP, 1:1000) overnight at 4°C.

Techniques: Labeling, Sequencing, Expressing, Western Blot