CUL3Δ403–459 displays increased auto‐ubiquitylation and ubiquitylation of KLHL3 A–DIn vitro ubiquitylation assays with purified proteins. CUL3Δ403–459 displays increased auto‐ubiquitylation and ubiquitylation of KLHL3 A–DIn vitro ubiquitylation assays with purified proteins. As ubiquitin is covalently attached to the substrate lysine residue, the appearance of higher molecular weight protein bands reflects the modification of the protein with ubiquitin. All assays contain purified UBE1, UBE2D3, ubiquitin, 0.1 mM ATP, 1 mM MgCl2 and were buffered in 50 mM HEPES, 150 mM NaCl and incubated at 30°C for the time indicated. Reactions were stopped by the addition of SDS–Laemmli buffer to a concentration of 1×. SDS–PAGE, staining with Coomassie blue, or detection with the indicated antibody following immunoblotting enabled the visualisation of ubiquitylation. (A) to determine the relative modification of KLHL3 by CUL3WT and CUL3Δ403–459, KLHL3 was included into the reaction at 2× molar concentration over CUL3WT and CUL3Δ403–459. (B, C) Reactions serve to determine basal auto‐ubiquitylation of CUL3WT or CUL3Δ403–459 and do not contain KLHL3 or other potential substrate proteins. Lysine residues on CUL3WT or CUL3Δ403–459 act as the substrate. (B) High molecular weight bands reflect ubiquitin chain linkages on CUL3 or the multiple mono‐ubiquitylation of a number of CUL3 lysine residues. (C) Activity assays contain methylated ubiquitin, a form of ubiquitin incapable of forming ubiquitin chains; as such, higher band shifts reflect the attachment of mono‐ubiquitin to one more lysine residue on CUL3WT or CUL3Δ403–459, respectively. (D) Activity assay performed as in (C) with methyl‐ubiquitin, the boxes shown on the gel are indicative of the gel pieces excised for mass spectrometry analysis in (F).ESchematic representation (to linear residue scale) highlighting the domains of CUL3 and schematic representation of the lysine residues of CUL3WT or CUL3Δ403–459, modified at 5 and 45 min, respectively, in the in vitro ubiquitylation assay shown in (D) and as determined by mass spectrometry.FStructural docking model of CUL3Δ403–459 based on the structure of full‐length CUL1 (1LDK) using Chimera (see Materials and Methods). The NTD is coloured green, while the CTD is red, and the four panels sample possible positioning of the CTD relative to the NTD in CUL3Δ403–459, assuming full flexibility of the linker after deletion of three helices encoded by exon 9. Source data are available online for this figure. Frances‐Rose Schumacher et al. EMBO Mol Med. 2015;7:1285-1306 © as stated in the article, figure or figure legend