Cathepsin d activity fluorometric assay kit

Protease activity was measured using commercially available kits (BioVision Cathepsin D Activity Fluorometric Assay Kit, #K143-100 and BioVision Cathepsin L Activity Fluorometric Assay Kit, #K142-100). Animals were staged as for immunoblotting, but without the addition of 20 mM FUDR. At day 1 of adulthood, worms were collected from plates with ice cold M9 and washed twice to remove food. Worm pellets were resuspended in 1% NP-40 buffer (Fisher Scientific) without protease inhibitors and frozen at -80 °C overnight. Pellets were thawed and sonicated for 4 cycles of 1 min on and 2 min off (BioRuptor, Diagenode). Lysates were centrifuged for 5 minutes at 13,000 rpm at 4 °C and supernatant was transferred to a fresh tube. 0.25 μg total protein per sample was used per assay and samples from one strain were run in triplicate. Fluorescence measurements were taken every minute at 25 °C (Infinite M200, Tecan). As controls, 250 nM Pepstatin A (for pan-aspartyl protease inhibition in CTSD assay, BioVision) or 10 μM CA-074 (for Cathepsin B inhibition, EMD Millipore, #205530) and 10 μM CTSLiII (for Cathepsin L inhibition, EMD Millipore, #219426) were added to the lysate and pre-incubated for 10 minutes on the bench at room temperature. Linear regression was performed on at least 30 minutes of data to calculate the rate of enzyme activity.

As previously described (Riessland et al., 2019 (link)), Cathepsin D enzymatic activity was assessed using the Cathepsin D Activity Fluorometric Assay Kit (BioVision) according to the manufacturers protocol. Values were then normalized to protein content using a BCA assay as described above.

The cathepsin D assay was performed using a fluorometric cathepsin D activity assay kit from Abcam (ab65302) according to the manufacturer's instruction. Briefly, cells were harvested and then dissolved with the cell lysis buffer provided in the kit. Protein concentration was determined by the BCA method; 50 ng of protein was incubated with the cathepsin D substrate mixture at 37°C for 1 h. Fluorescence intensity of the synthetic substrate treated with cathepsin D was measured using a fluorescence plate reader (Infinite M200 PRO, Tecan) at Ex/Em = 328/460 nm.

Cathepsin D activity was measured using a Fluorometric cathepsin D activity assay kit (Abcam, Cambridge, MA).

Lysosome-enriched fraction and cytoplasmic fraction were prepared as described above in glucocerebrosidase activity assay. The lysosome-enriched fraction was obtained from the pellet after centrifugation at 17,000 X g for 10 minutes, and the supernatant was used as the cytoplasmic fraction. Equal amounts of protein (15 μg) were used for the cathepsin D activity assay. Fluorescence values of these samples were measured using cathepsin D Activity Assay Kit/Fluorometric (Abcam, ab65302), following the manufacturer’s protocol. The actual enzyme activities were then calculated using a standard curve of the substrate by cathepsin D (C3138, Sigma).