Figure 2
From: Unique E2-binding specificity of artificial RING fingers in cancer cells
Substrate-independent assays of artificial RING finger (ARF) ubiquitination in vitro. ARF mutants ((A) P31A, (B) K32A, (C) L33A, (D) T34A, and (E) C35A) were incubated with randomly biotinylated ubiquitin (Ub), recombinant E1, and 11 E2s. ARF mutants promoted the addition of Ub chains to itself. The reactions were resolved using NuPAGE. Ub chains on PVDF membranes were visualized using Vectastain ABC Elite Kits. Emitted signals were detected using WSE-6100 LuminoGraph I.
