Quantification of Lysosomal Hydrolase Activity

Cathepsin D activity (used as a proxy for total lysosomal hydrolase activity) was measured by using Cathepsin D Activity Assay Kit (Abcam) as previously described^{35 (link)}. Cells were collected, and 350 μL of lysis buffer was used to lyse 1 million cells; 5 μL of lysate was incubated in substrate/buffer solution for 75 min at 37 °C. Enzyme activity was measured by monitoring release of the fluorescent cleavage product, MCA. Activity measurements from parallel reactions containing 0.7 μM protease inhibitor pepstatin A were subtracted from the activity measurements obtained without the inhibitor. Fluorescence was quantified by SpectraMax M5 at Ex/Em = 328/460 nm.