Cathepsin d activity assay

CTSD activity was measured by Cathepsin D Activity Assay (Abcam) according to the manufacturer’s protocol. Briefly, CD8⁺ T_eff cells (1 × 10⁷) were lysed with 300 µl of chilled CD Cell Lysis Buffer and centrifuged at 21,000g for 5 min at 4 °C to collect the supernatant. In total, 50 µl supernatant and 52 µl reaction mix (50 µl reaction buffer and 2 µl substrate) were added to the black 96-well plate and incubated for 30 min at 37 °C. The fluorescence released upon substrate cleavage was measured using Synergy H1 microplate reader at Ex/Em = 328/460 nm. Cathepsin D activity was shown by the RFUs per microgram of protein of samples.

Cathepsin D activity was determined using a fluorescence-based assay “Cathepsin D activity assay” from Abcam. This assay utilizes the preferred cathepsin-D substrate sequence GKPILFFRLK(Dnp)-D-R-NH2) labelled with MCA. 0.1–10 μg of protein were used per assay according to manufacturer’s instructions. Assay plates were incubated at 37 °C and readings were performed in a fluorometer at 355 nm excitation and 520 emission for 2 hours. Results are expressed as a slope of fluorescence emission after 2 hours per μg of protein.