Fig. 1. NR is a small-molecule inhibitor of USP4 activity. (A) Chemical structure of NR. (B) The DUB activity of USP4, YOD1, USP5, UCHL1, and USP14 with and without 200 µM of NR was compared using Ub-AMC deubiquitination assays in which AMC hydrolyzed from the Ub-AMC substrate was measured as fluorescence at an excitation wavelength of 336 nm and an emission wavelength of 441 nm. (C) The effect of various concentrations of NR on the deubiquitinating activity of USP4 was measured using Ub-AMC deubiquitination assays. Error bars represent SD, ***p<0.001, ****p<0.0001. (D) The effect of various concentrations of NR on the deubiquitinating activity of USP4 was measured by monitoring the band shift from GST-Ub-HA to GST-Ub. (E) The effect of various concentrations of NR on the deubiquitinating activity of USP4 was measured by monitoring Ub cleavage from K48- (left) and K63- (right) linked polyubiquitin chains. The activity was examined on 15% SDS-PAGE gel with Coomassie Blue staining. (F) Michaelis-Menten plot for calculating the DUB activity of USP4. Hydrolysis Ub-AMC assays were performed after USP4 reacted with Ub-AMC (0-225 nM) in the presence of NR at various concentrations (0-15 µM).