Cathepsin S enzyme activity assay

Cathepsin S activity assay kit (Fluorometric) was purchased from Abcam (Cambridge, MA). The cells were collected and lysed on ice in cathepsin S cell lysis buffer for 10 min. The lysates were centrifuged at 13,000 x g for 5 min at 4°C, and the supernatant fractions were collected. Assay were performed in 96-well microtiter plates by incubation 50 μg of cell lysates in 50 μl of reaction buffer containing a 200 μM cathepsin S substrate (Ac-VVR-AFC). Lysates were incubated at 37°C for 1 h. The enzyme activity was measured with a fluorometric plate reader at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Fold-increase in cathepsin S activity was determined by comparing the relative fluorescence units (RFU) with the level of the control sample.