19s proteasomes

19S proteasomes (PA700, Boston Biochem) were pre-treated with Ub-VS (ubiquitin vinyl sulfone, Boston Biochem), an inhibitor of deubiquitinating enzymes, at 30 °C for 2 h to block the associated deubiquitinating activity. USP14 (Enzo) was mixed with the pre-treated 19S proteasomes or proteasome-enriched cell lysates, along with other purified proteins as indicated, in Ub-AMC assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM EDTA, 1 mM ATP, 1 mM DTT, and 1 mg ml⁻¹ BSA). The reaction was initiated upon the addition of Ub-AMC (Boston Biochem), and the fluorescence of released AMC was monitored on a microplate fluorometer (Infinite M200, Tecan), using 345 nm and 445 nm wavelengths of excitation and emission, every 3 min at 37 °C.

In vitro proteasome activity assays were performed using total cellular extracts of OPM-2 cells (25 μg) in reaction buffer (25 mM Hepes, 0.5 mM EDTA, 0.03% SDS). We used the substrates Suc-LLVY-2R110 (β5c), Z-LLE-2R110 (β1c) and (Ac-KQL)2R110 (β2c) from AAT Bioquest (Sunnyvale, CA). A Tecan Infinite 200 reader equipped with 498 nm excitation and 520 nm emission filters was used for detection. For DUB inhibition assays, Ubiquitin-Rhodamine 110 (Rh110) (Boston Biochem, Cambridge, MA) was used as a substrate and either total cell lysates or 19S proteasomes (Boston Biochem) as source of enzyme using procedures described previously^{28 (link)}. Labeling with ubiquitin-vinylsulphone was performed as described^{64 (link)} using reagent from Boston Biochem.