Fig. 1: A suite of synthetic antibody fragments (Fabs) specific for NEDD8-modified cullin-RING ligases (CRLs).

a, CRLs are switched on by site-specific NEDD8 linkage to the cullin's WHB domain. Neddylation promotes the active conformation required for ubiquitylation to be assumed (SBM = substrate binding module, UCE = ubiquitin carrying enzyme). b, Strategy to select Fabs specifically binding to the NEDD8-modifed, and not unmodified, CRLs. Selections were performed with neddylated C-terminal regions of CUL1 or CUL2 bound to RBX1. c, Binding specificity of Fabs developed with selection strategy seen in (b) against non-neddylated and neddylated CUL1-5, GST and BSA as determined by ELISA. Baits used for selection of specific Fabs are indicated. d, Immunoblot using indicated Fabs of the suite as primary binders for recognition of indicated purified recombinant cullins modified by NEDD8 (+) or not (−). e, IP with indicated Fabs from K562 (human lymphoblast) cells treated with DMSO or MLN4924, followed by immunoblotting against cullins 1-5. Slower migrating forms of cullins eliminated by MLN4924 treatment are interpreted as NEDD8-modified, whereas faster-migrating forms of cullins are interpreted as unneddylated. * - band cross-reacting with anti-CUL4 antibody. f, Dose-response curve of MLN4924 in K562 cells as measured by flow cytometry using N8C_Fab3b fluorescently labeled with Alexa Fluor 647 as a direct read-out of cullin neddylation levels.