Figure 3. Mutations to Lys⁵, Lys⁷ and Lys¹¹ of the N-terminal SUMO-2 do not affect ubiquitylation by Ube2W and RNF4.

(A) Schematic presentation of isopeptide linked-poly-SUMO-2 with the first SUMO unit being full length, and the remaining three units lacking 11 N-terminal amino acids [Pep.6His-SUMO-2_×1–SUMO-2-(12–92)_×3]. This was used to create lysine residue mutants for biochemical analysis. (B) Four mutants of the first SUMO-2 were created, and the constructs analysed by in vitro ubiquitylation. Reactions were incubated for 30 min and products were visualized by Coomassie Blue-stained SDS/PAGE. MW, molecular mass; Ub, ubiquitin; wt, wild-type.