Fig. 2: Effects of neddylated cullin targeting Fabs on CRL activities.

a, CSN-catalyzed NEDD8 deconjugation is visualized by accumulation of free NEDD8 observed over time by SDS-PAGE. Assays test protective effects of incubating 10x molar excess of indicated Fabs with neddylated C-terminal regions of CUL1 or CUL2 complexes with RBX1. b, Effects of N8C_Fab3b binding on ubiquitylation efficiency of neddylated CRL1^BTRC-UBE2D (aka CRL1^βTrCP1-UBE2D) as determined by a qualitative substrate priming assay monitoring transfer of fluorescent ubiquitin (*Ub) to a phospho-NFKBIA (aka phospho-IκBα) derived substrate peptide (pNFKBIA^PEP) by SDS-PAGE. c, As (b), but monitoring the transfer of *Ub by neddylated CRL1^FBXW7-UBE2L3/ARIH1 to a phospho-CCNE (aka phospho-Cyclin E) derived substrate peptide (pCCNE^PEP). d, As (b), but monitoring Pomalidomide induced transfer of *Ub by neddylated CRL4^CRBN-UBE2D to the IKZF ZF2 substrate. e, As (b), but monitoring Pomalidomide induced transfer of *Ub by neddylated CRL4^CRBN-UBE2L3/ARIH1 to the IKZF ZF2 substrate. f, As (c), but comparing *Ub transfer in the absence and presence of CSN with or without prior incubation with N8C_Fab3b.