Fig. 1: Activity of circulating proteasomes (c-proteasomes) in human plasma.

a Plasma samples were collected from four individuals (plasma A–D) in EDTA tubes, and their c-proteasome activity (in 20 μL of plasma) was evaluated by monitoring the hydrolysis of the fluorogenic reporter substrates (final concentration of 250 μM in a total of 100 μL reaction), such as suc-LLVY-AMC (for chymotrypsin-like activity), Boc-LRR-AMC (for trypsin-like activity), and Z-LLE-AMC (for caspase-like activity) in the presence or absence of the proteasome inhibitor MG132 (10 μM). These reactions were performed using assay buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mg/mL BSA, 1 mM ATP, and 1 mM DTT). Sodium dodecyl sulfate (SDS) was not added to the reaction unless otherwise described. The graphs (left) represent the results obtained in three independent experiments, and the mean of the raw fluorescence values (right) at 60 min are plotted with their standard deviations (N = 3). b Human c-proteasome activity was analyzed using suc-LLVY-AMC as the substrate, along with a wide range of protease inhibitors, including aprotinin (trypsin inhibitor), pepstatin A (aspartyl protease inhibitor), and leupeptin (serine/cysteine protease inhibitor). c As in (b), but using different proteasome inhibitors (10 μM MG132, 2 μM bortezomib, 2 μM epoxomicin, or 100 nM carfilzomib). d The plasma samples were preincubated with SDS at the indicated final concentrations for 10 min before initiating the suc-LLVY-AMC hydrolysis reactions. Relative fluorescence values after 30-min reactions were normalized to those obtained in the presence of 10 μM MG132. The values represent the mean ± standard deviation (N = 3). e As in (a), except that assay buffer without ATP was used. No significant changes were observed.