Quantitative Proteasome Activity Assay

Frozen cell pellets were lysed in a buffer of 50 mM Tris–HCl, pH 7.5, 25% sucrose, 2 mM EDTA, 1 mM DTT, 1 mM ATP, 0.05% digitonin. Proteasome activity was measured in extracts of treated cells with Suc-LLVY-amc (ß5c and LMP7), Ac-ANW-amc (LMP7), Ac-WLA-amc (ß5c), Ac-nLPnLD-amc (ß1c and LMP2), Ac-APL-amc (LMP2) and Ac-RLR-amc (ß2c and MECL1) fluorogenic substrates^{36 (link)}. It was normalized to the total protein content of the extract, which was determined using the Coomassie Plus—The Better Bradford Assay Reagent (Thermo). To distinguish between contribution of ß5c and LMP7 to the cleavage of Suc-LLVY-amc, extracts were preincubated with a highly LMP7-specific inhibitor LU-015i (5 μM) for 30 min at 37 °C immediately before activity measurements. Alternatively, occupancy of active sites was measured with activity-based probes as described^{10 (link),37 (link)}. In a three-probe-cocktail, ß1c (PSMB6) and LMP2 (ß1i) subunits are labeled with Cy5-NC-001, BODIPY(FL)-LU-112 labels MECL1 (ß2i) and ß2c (PSMB7), and LMP7 (ß5i) and ß5c (PSMB5) subunits were labeled with BODIPY(TMR)-NC-005-vs^{10 (link)}. In a two-probe combination, LMP7, ß5c, MECL1 and ß2c subunits were labeled with BODIPY(TMR)-NC-005, and ß1c and LMP2 were labeled with BODIPY(FL)-NC-001^{37 (link),47 (link)}. Labeled subunits were resolved in 10% Bis–Tris SurePAGE™ (Genscript) and imaged on c600 gel imager (Azure Biosystems).