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. 2019 Dec 19;23(1):100790. doi: 10.1016/j.isci.2019.100790

Figure 1.

Figure 1

Proteasome Inhibition Disrupts the Association of USP14 with PSMD2 in the Proteasome 19S Subunit

(A) SH-SY5Y cells overexpressing Flag-WT-USP14 were treated with DMSO or lactacystin (5 μM, 6 h) and subjected to immunoprecipitation. The complexes were analyzed by immunoblotting for PSMD2 of the proteasome 19S regulatory particle and Flag. Densitometry ratio of the PSMD2-USP14 interaction is presented in the right panel.

(B) Protein sequence of USP14 UBL domain is conserved among species.

(C) Tryptophan (W) mutated to alanine (A) in W58A-USP14 sequence is underlined.

(D) Immunoprecipitation of Flag-WT-USP14 or Flag-W58A-USP14 constructs with Flag antibody and analyzed by immunoblotting for PSMD2 and Flag.

(E) Cell lysates of Neuro2A cell overexpressing Flag-WT-USP14, Flag-C114A-USP14, or Flag-W58A-USP14 were incubated with the Ub-VME substrate for 3 h to assess DUB activity. The mixture was analyzed by immunoblotting for Flag, to detect the substrate-bound DUBs (shows as an additional band on the IB, marked by an arrow; star marks the overexpressed Flag constructs).

Abbreviations: USP14, ubiquitin-specific protease-14; WT, wild-type; PSMD2, 26S proteasome regulatory subunit 2; UBL, ubiquitin-like; Ub-VME, ubiquitin-vinyl methyl ester; DUB, deubiquitinating enzyme; IB, immunoblotting; IP, immunoprecipitation. (A) n = 3, **- p ≤ 0.01 Student's t-test.