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. 2012 Sep 22;68(Pt 10):1158–1163. doi: 10.1107/S1744309112036937

Figure 3.

Figure 3

Formation of the ubiquitin thioester. Top: in vitro formation of the ubiquitin-thioester intermediate with the UBR5 HECT C-lobe in the presence of UBE1, UBCH4 and ubiquitin (Ub) shows that the UBR5 C-lobe is catalytically active. Mutation of the UBR5 catalytic cysteine to alanine (mt) abolishes the formation of the C-lobe–ubiquitin intermediate. The products were resolved by SDS–PAGE and visualized using Coomassie Blue. Bottom: disruption of the thioester C-lobe–ubiquitin bond in the presence of 50 mM DTT.