Cathepsin d activity assay kit

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.

Measurement of Pep4/Cathepsin D activity was performed using a fluorometric Cathepsin D activity assay kit from Abcam (ab65302) according to the manufacturers protocol. For analysis of Pep4 activity in yeast, 2x10⁶ cells were harvested 16 h after induction of galactose-driven expression of αSyn. Protein extracts were generated by mechanical lysis using glass beads and the supplied CD cell lysis buffer. For measurement of Cathepsin D activity in D. melanogaster, five fly heads per sample were collected at indicated time points and subsequently mechanically lysed in supplied CD cell lysis buffer. Protein concentration was determined via Bradford assay (Bio-Rad) and 0.1 μg protein was used for the Pep4/Cathepsin D activity assay. Reactions for yeast samples were incubated for 2 h at 28°C and fly samples for 2 h at 25°C. Fluorescence signal was measured with a Tecan Genios pro microplate reader (ex. 328 nm, em. 460 nm). Of note, lysates from Δpep4 yeast strains or wild type flies treated with 150 μM pepstatin A (dissolved in DMSO) were used as background control.

Cathepsin D activity was determined using the Cathepsin D activity assay Kit (Abcam). Each channel of the Brain-Chip was lysed in 200 µl chilled CD cell lysis buffer supplied in the kit. Cells and the reaction mix were prepared according to manufacturer’s protocol. The fluorescence derived from cathepsin D-mediated substrate cleavage was measured using a BioTek plate reader at excitation/emission = 328/460 nm.

Cathepsin D activity assay kit (ab65302, Abcam) was used to quantify the activity of Cathepsin D in cell lysates. The procedure was performed according to the manufacturer’s protocol.

Cathepsin D activity was determined using Cathepsin D activity assay kit from Abcam as previously described26 (link). Results are expressed as a slope of fluorescence emission after 1 h per μg of protein.