Fig. 3: CatD-P3 activation in vitro.

a Time-dependent activation of CatD-P3 (20 μM) in reaction buffer in the presence or absence of CatD (50 nM). Ctrl = probe alone. b CatD (50 nM) induced increase in the fluorescence intensity of CatD-P3 (20 μM) with and without the inhibitor Pepstatin A (20 µM) (n = 3). c Activation of CatD-P3 in reaction buffer and in pH 4 adjusted 10% FBS over 1 h. Fluorescence signal amplification was comparable under both conditions, with slightly higher background fluorescence in the 10% FBS media. d The graph represents the assessment of probe CatD-P3 specificity for cathepsin D in comparison to other proteases associated with the immune response. CatD-P3 (20 µM) was incubated with each protease (at a concentration of 5 nM) in their respective optimal reaction buffers for 1 h. To quantify the relative cleavage levels, the fluorescence increases were normalised to a maximum fold change of 1, which corresponds to the maximum cleavage achieved by cathepsin D. This normalisation was performed by dividing the fluorescence reading of each protease-activated probe by the maximum fluorescence value (RFU_max) obtained in the corresponding buffer for fully cleaved CatD-P3 (20 µM), as detailed in the methods section. Measurements were taken in triplicate from distinct samples and mean values and standard deviation error bars were calculated and plotted using OriginLab.