Versafluortm fluorometer system

Proteasome peptidase activities in human skin fibroblasts were measured at a VersaFluor^TM Fluorometer System (Bio-Rad laboratories, Hercules, CA, USA) as described previously^{64 (link)} by using the Boc-Leu-Arg-Arg-AMC-LRR (trypsin-like activity, T-L), Z-Leu-Leu-Glu-AMC-LLE (caspase-like activity, C-L) and Suc-Leu-Leu-Val-Tyr-AMC-LLVY (chymotrypsin-like activity, CT-L) (Enzo Life Sciences, Inc. Farmingdale, NY, USA) fluoropeptides.

The chymotrypsin-like activity in young BJ skin fibroblasts was evaluated, as described previously [33 (link)]. Briefly, the hydrolysis of the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC (Enzo Life Sciences, Inc., NY, USA) was recorded in a Versa Fluor^TM Fluorometer System (Bio-Rad laboratories, Hercules, CA, USA) at excitation and emission wavelengths of 360 and 440 nm, respectively. The concentration used for each extract is mentioned in Table 1.

Measurement of proteasome proteolytic activities was performed as previously described [22 (link),56 (link)]. Briefly, human fibroblasts were plated in 60-mm dishes and were treated for 24 h with a concentration of 5 μM for each compound and 1 μg/mL and 10 μg/mL of concentration for each extract. After the treatment cells were lysed with a buffer suitable for the isolation of 26S proteasome (0.2% Nonidet P-40, 5 mM ATP, 10% glycerol, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20 mM Tris, pH 7.6). Lysates were cleared with centrifugation at 19,000g (4 °C) and 20 μg of proteins were immediately used to determine the main proteasome proteolytic activities [chymotrypsin-like (CT-L/LLVY) and caspase-like (C-L/LLE)]. Activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc–Leu–Leu–Val–Tyr–AMC and Z-Leu–Leu–Glu–AMC (Enzo Life Sciences), at 37 °C for 30 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories) at excitation and emission wavelengths of 350 and 440 nm, respectively. Each sample was prepared in duplicate.