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. Author manuscript; available in PMC: 2021 Dec 3.
Published in final edited form as: Mol Cell. 2020 Nov 5;80(5):796–809.e9. doi: 10.1016/j.molcel.2020.10.017

FIGURE 1. UCH37 Cleaves K48 Linkages in Branched Ubiquitin Trimers.

FIGURE 1.

(A) Structures of native and thiol-ene coupling (TEC)-derived isopeptide bonds.

(B) SDS-PAGE analysis of cleavage reactions with TEC-derived branch tri-Ub derivatives. Concentrations used: tri-Ub (10 μM); UCH37 (1 μM). Linkages in branched tri-Ub are represented by X/Y, where X and Y denote the positions of the isopeptide bonds.

(C) SDS-PAGE analysis of K6/K48 branch tri-Ub (10 μM) cleavage with varying concentrations of UCH37 (0, 0.1, 0.25, 0.5, 1, 5, and 10 μM) at 1 h.

(D) SDS-PAGE analysis of the cleavage reaction time course. Concentrations used: native tri-Ub (10 μM); UCH37 (1 μM).

(E) Sortagging native K6/K48 branch tri-Ub with different fluorophores to report on the linkage specificity of UCH37.

(F) Fluorescence analysis of cleavage reactions with either OTUB1 (5 μM) or UCH37 (5 μM) and fluorophore-labeled native K6/K48 branch tri-Ub (10 μM).

See also Figure S1.