Ubiquitin rhodamine 110 kinetic assay

DUBs from the high-throughput screen were tested for their respective activity in the Ub-Rho110 assay in the presence or absence of inhibitors using Nunc™ 384-well plates (Thermo Scientific™ catalog number: 2262260). Individual DUBs were pre-incubated with different concentrations of inhibitors in assay buffer (50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween-20, 5 mM DTT). Compounds and protein were incubated for 30 minutes at room temperature prior to the addition of Ub-Rho110 (Boston Biochem) substrate. The initial rate of the reaction was measured by collecting fluorescence data at one-minute intervals over 30-minute period using a Clariostar fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm respectively. The calculated initial rate values were normalized to no compound control wells and were plotted against inhibitor concentrations to determine IC₅₀s. All the experimental data were plotted using GraphPad Prism (log(inhibitor) vs. normalized response - Variable slope, least squares fit). All assays were performed at least twice for each compound and error bars are reported as SEM.