Fig. 3.

Characterization of CTSD activation by ADAM30. (a) Proteolysis of the pro-CTSD for CTSD activation. The black arrow shows the cleavage site for ADAM30 between amino acids 167 and 168 (corresponding to the activational processing of CTSD). (b) In vitro measurement of CTSD activity (mean ± SD) after incubation of recombinant BSA, ADAM30^WT or ADAM30^mut with a CTSD peptide substrate. (c) Proximity ligation assay with human endogenous ADAM30^WT and CTSD proteins in native SKNSH-SY5Y and with overexpressed ADAM30^WT and endogenous CTSD in HEK293 cells. CTSD activity in HEK293-APP695^WT (d) or SKNSH-SY5Y-APP695^WT (e) cells transfected with ADAM30^WT, ADAM30^mut or empty vector (Mock). The CTSD activity was assayed in three independent experiments, with a measurement every at 5, 10 and 15 min (means ± SEM). Top-right: representative kinetic experiment. (f) CTSD activity in SKNSH-SY5Y cells transfected with ADAM30-siRNA. The CTSD activity was assayed in three independent experiments, with a measurement every at 5, 10 and 15 min (means ± SEM). Top-right: representative kinetic experiment.

*p < 0.05 (Mann-Whitney non-parametric test). Three independent experiments were performed in duplicate.