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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 4. UCH37 Preferentially Binds K48 Chains.

FIGURE 4.

(A) SDS-PAGE analysis of pull-downs with M1-, K6-, K11-, K29-, K33-, K48-, and K63-linked Ub chains. Each chain (50 pmol) was captured using immobilized His-RPN13•UCH37 C88A (5 nmol).

(B) Schematic for measuring the hydrolysis of native K6/K48 branched tri-Ub in the presence of K48 tetra-Ub added in trans.

(C) Michaelis-Menten plot for the hydrolysis of native K6/K48 branched tri-Ub by UCH37•RPN13 (0.5 μM) in the presence of K48 tetra-Ub.

(D-E) ITC analysis of active UCH37•RPN13 binding to K48-linked tri-Ub (C) and tetra-Ub (D).

(F) Schematic for measuring the hydrolysis of native K6/K48 branched tetra-Ub in cis.

(G) Michaelis-Menten plot for the hydrolysis of native K6/K48 branched tetra-Ub by UCH37•RPN13 (0.5 μM).

All ITC curves are representative traces and reported Kds (B, C) are derived from averaging fits of 2 independent experiments. All kinetic curves (A, D) are representative traces and constants derived from averaging fits of 2 independent experiments with SD. See also Figure S4 and Table S1D.