Ub vme

Fluorescent enzymatic assays were performed by following the hydrolytic liberation of AMC from ubiquitin-AMC (Ub-AMC, Boston Biochem) or LRGG-AMC. Measurements were performed in parallel in a final volume of 100 μL using 96-well ½ area black plates with a 2300 Multilabel Reader (PerkinElmer). Assay results were analyzed with Prism 5.0 (GraphPad). For the Ub-vinyl Methyl Ester (Ub-VME, Boston Biochem) conjugation assays, proteins were incubated with Ub-VME at a molar ratio of 1:2 before SDS-PAGE analysis. See Supplemental Information for details.

Deubiquitinylation activity of recombinant Uch-L1 was fluorometrically assayed using Ub-7-amido-4-methylcoumarin (Ub-AMC, Boston Biochem) as described (29 (link)) with minor modification. In brief, 0.25 μM Ub-AMC and 0.02 μM Uch-L1 (or SNO-Uch-L1) were mixed in a final volume of 100 μl in reaction buffer (50 mM Tris-HCl, pH 7.6, 0.5 mM EDTA), and incubated for 10 min at 25 °C. The amount of AMC released from the Ub-AMC substrate was monitored using SpectraMax M2 (Molecular Devices) with excitation and emission wavelengths of 380 nm and 460 nm, respectively.
Activity of Uch-L1 in cells or tissue lysates was assessed with the activity-based probe, ubiquitin-vinylmethyl ester (Ub-VME), irreversibly modifying the “active” form of ubiquitin C‐terminal hydrolases and thus causing a shift in electrophoretic mobility on immunoblots (55 (link), 56 (link)). For the Ub-VME assay, cell or tissue lysate was freshly prepared in PBS containing 0.1% Triton X-100. The reaction was initiated by incubating 2 μg of lysate with 0.2 μM Ub-VME (Boston Biochem), incubated for 5 min at room temperature, and stopped by adding NuPAGE LDS sample buffer (Thermo Fisher Scientific). Proteins were resolved by NuPAGE Bis-Tris gel followed by western blotting.