Coomassie plus the better bradford assay reagent

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).

The amount of procollagen type I in cell lysates and secreted by fibroblasts was measured using Human pro-Collagen I alpha 1 Simple Step Elisa Kit (Abcam, Cambridge, UK). After 24 h treatment of cells with tested compounds, the experimental media were collected and centrifuged at 2000× g for 10 min. The concentration of protein in cell culture supernatants was determined with Coomassie Plus—The Better Bradford Assay Reagent (Thermo Fisher Scientific, Rockford, IL, USA). Cells were washed three times with PBS and harvested with Extraction buffer provided with the assay. After incubation on ice for 20 min samples were centrifuged at 18,000× g for 20 min at 4 °C. The concentration of protein in cell lysates was determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). A standard curve was prepared using Procollagen I alpha 1 standard in the concentration of 10–1000 pg/mL. Aliquots of samples (50 µL/well) of an appropriately diluted cell lysates and experimental media, containing 0.5–1 mg of total proteins, were added to a 96-well microtitre plate coated with procollagen type I standard and then the manufacturer’s protocol was followed. The assays were done in duplicates in three independent experiments. The secretion of procollagen was calculated by dividing the amount of procollagen released into the cultured medium by the sum of medium and intracellular collagen.