Ab65306

The relative activities of Cathepsin B (CTSB), Cathepsin D (CTSD), and CTSL were measured, respectively, using Cathepsin Activity Assay Kits (Fluorometric) (ab65300, ab65302, and ab65306; Abcam) following the manufacturer’s instructions. The CTSB assay kit employs a specific CTSB substrate sequence RR labeled with AFC. Consequently, the cleavage of the synthetic substrate RR-AFC and the subsequent release of free AFC can provide an indication for the CTSB activity. The CTSD assay kit utilizes a preferred CTSD substrate sequence GKPILFFRLK(Dnp)-D-R-NH2 labeled with MCA. The resulting fluorescence released from the substrate can reflect the level of CTSD activity. The CTSL assay kit utilizes a preferred CTSL substrate sequence FR labeled with AFC, with the released free AFC as an indicator of the CTSL activity. Briefly, 1 × 10⁶ astrocyte cells overexpressing pcDNA3.1-A53T-SNCA or exposed to aggregated α-syn were harvested and washed with 200-μl ice-cold PBS. The cells were resuspended in 200-μl chilled CD Cell Lysis Buffer on ice for 10 min and centrifuged at 12,000×g to remove large cell debris. Cell lysate (50 μl/well) was transferred into a 96-well plate and incubated with 50 μl of reaction buffer and 2 μl of substrate at 37 °C for 1 h. Samples were then quantified using a fluorescence plate reader (FlexStation 3, Molecular Devices, San Jose, CA) at Ex/Em = 328/460 nm.