Measuring Cathepsin B and L Activity

Cathepsin B, L activity was measured as described previously [54 (link),55 (link)] with minor modifications. Human foreskin fibroblasts (BJ) were plated in 60-mm dishes and were treated for 24 h with a concentration of 1 μg/mL and 10 μg/mL of concentration for each extract and 5 μM for each compound. Cells were lysed on ice in 1 mM dithiothreitol and 50 mM Tris, pH 4.0 and the lysates were cleared at 14,000×g for 20 min at 4 °C. Following protein content measurement with Bradford assay (Bio-Rad laboratories, Hercules, CA, USA), 20 μg of protein were incubated in the reaction buffer (50 mM sodium acetate, 8 mM cysteine-hydrochloride, 1 mM EDTA, pH 4.0) containing the substrate z-FR-AMC (Enzo Life Sciences, Farmingdale, NY, USA) for 30 min at 37 °C. The fluorescence was measured (VersaFluorTM Fluorometer System, Bio-Rad laboratories) at excitation and emission wavelengths of 350 nm and 440 nm, respectively.