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Kinetics of Ub-N41 cleavage in the proteoliposome assay. (A) Ligation of ubiquitin–thioester to N41 notch peptide. (B) Purified Ub-N41 and N43 visualized via their C-terminal fluorescein tag. (C) After 18 h, Ub-N41 cleavage product was monitored by running proteoliposome reactions on a 16.5% Tris–tricine gel and imaging with a fluorescence scanner. Cleavage was greatly reduced compared with N43 (compare with Fig. 3E), and saturating concentrations were not achieved (n = 2).
