2.8. Furin activity assay

Serial dilutions of AT were mixed with recombinant furin (R & D; 1503‐SE‐010) in assay buffer (25 mM Tris, 1 mM CaCl₂, pH 9) for 10 min at 37°C, followed by addition of 50 µl of 20 µM succinyl (Suc.) modified QTNSPRRAR‐AMC protease substrate. The final concentration of furin was 1.5 ng/µl. Fluorescence intensity was recorded at 2‐min intervals for 5 h at an excitation wavelength of 355 nm and emission wavelength of 460 nm at 37°C in a Cytation 3 (BioTek) microplate reader. The area under the curve values normalized to furin control was plotted. The activity of activated AT on furin was assessed as described above, using activated AT.