Extended Data Figure 8. Qualitative validation of mechanistic principles underlying substrate priming by neddylated CRLs and UBE2D.

Gel panels in this figure are representative from two independent experiments; n=2 technical replicates.

a, Schematic of a qualitative substrate priming assay for testing effects of mutations in neddylated CRL1^β-TRCP or UBE2D on substrate priming, monitoring fluorescent UB transfer from UBE2D3 to the phosphorylated IκBα substrate. b, Scan of SDS-PAGE detecting fluorescent UB transferred to IκBα-derived substrate in qualitative assay for NEDD8 activation of substrate priming. c, as in b, showing effect on substrate priming of disrupting the activation module with the NEDD8 Q40E mutation. d, as in b, showing the effect on substrate priming of disrupting interactions between the activation and catalytic modules with NEDD8 I44A or UBE2D S22R mutation. e, as in b, showing the effect on substrate priming of disrupting interactions between the activation and substrate scaffolding modules, though CUL1 modification by a “Ubiquitylized” NEDD8 mutant with six residues swapped for UB counterparts (L2Q K4F E14T D16E G63K G64E). f, as in b, showing the effect of UBE2D H32A mutation at the interface between the catalytic and substrate scaffolding modules. g, Scheme of pulse-chase assay for testing effects of mutations in neddylated CRL1^FBW7 or UBE2D on substrate priming. Assay monitors transfer of fluorescent UB from UBE2D to peptide substrate derived from phosphorylated Cyclin E (pCyE). h, Fluorescent scan detecting UB transferred to the pCyE substrate by neddylated CRL1^FBW7 and the indicated mutants of UBE2D. i, Fluorescent scan detecting UB transferred to the pCyE substrate by UBE2D and indicated variants of neddylated (or ubiquitylated) CRL1^FBW7. Experiment with unneddylated CRL1^FBW7 use the K720R variant of CUL1 to prevent artifactual ubiquitylation. j, Scheme of pulse-chase assay for testing effects of mutations in neddylated CRL4^CRBN or UBE2D on substrate priming, monitoring fluorescent UB transfer from UBE2D to the IKZF1/3 ZF2 substrate in the presence of the immunomodulatory drug pomalidomide. k, Fluorescent scan of assay validation, showing pomalidomide-dependence. l, Fluorescent scan detecting UB transferred to the IKZF substrate by CRL4^CRBN, pomalidomide and the indicated variants of UBE2D. m-o, Fluorescent scan detecting UB transferred to the IKZF substrate by UBE2D and the indicated variants of neddylated (or ubiquitylated) CRL4^CRBN with pomalidomide. Experiments with unneddylated CRL4^CRBN use the K705R variant of CUL4A to prevent artifactual ubiquitylation.