Furin inhibitor 2

SEMA3G cDNA was purchased from Horizon Discovery MGC cDNA (cat# MHS6278‐211689602) and PCR‐amplified containing a C‐terminal 3xflag sequence. The transgene was subsequently subcloned into pCDNA3 and the sequence verified before being transiently transfected (Invitrogen Lipofectamine 2000) into HEK 293T cells as per the manufacturer's protocol for 10 cm tissue culture dishes. Twenty‐four hours after transfection, cells were cultured on Opti‐MEM for 5  days in the presence of 100 μM Furin Inhibitor II (Sigma, SCP0148‐5MG) and media were collected daily and stored at 4°C. Media were then pooled, buffer exchanged into 1× PBS pH 7.4 (Gibco, cat# 10010023), and concentrated 10× fold using a 50 kDa filter concentrator (Vivaspin500). SEMA3G containing conditioned media was validated by western blot analysis using anti‐Flag antibody (Sigma, cat# F1804) as well as anti‐SEMA3G antibody (Invitrogen, cat# PA5‐51631) at the manufacturer's recommended dilutions.

A volume of 10 µl of sera was preincubated with 1 of 9 protease inhibitors included in a commercially available protease inhibitor set (Roche) for 30 min at 30°C. Individual inhibitors (including antipain, bestatin, chymostatin, E-64, phosphoramidon, Pefabloc SC, and aprotinin) were reconstituted per the instructions of each manufacturer. Following this incubation, 1.5 µg of rPA was added to the mixture and incubated at 37°C for 1 h. Specific inhibition of furin activity was accomplished using furin inhibitor I (Cayman Chemical Company) and furin inhibitor II (Sigma). These compounds are selective competitive inhibitors of the proprotein convertases, including furin. Serum (12 µl) was incubated with furin inhibitors at room temperature for 10 min, after which rPA (1.5 µg) was added and the entire mixture incubated for an additional 1 h at 37°C.