After centrifugation to remove insoluble debris and dead cells, cell culture medium was concentrated with the Amicon Ultra-0.5 30K Centrifugal Filter Devices (EMD Millipore, Darmstadt, Germany). The concentrate (25 μl) was then prepared in a buffer consisting of 31.3 mM Tris-HCl (pH 6.8), 1% wt/vol SDS, 5% glycerol, and 0.025% wt/vol bromophenol blue, and boiled for 5 min. Lysates from proximal tubular cells were prepared in a buffer consisting of 62.5 mM Tris⋅HCl (pH 6.8), 2% wt/vol SDS, 10% glycerol, 50 mM DTT, and 0.01% wt/vol bromophenol blue, boiled for 5 min, and centrifuged at 10,000g for 5 min at 4°C to remove insoluble debris.
Twenty-five micro liter of concentrated media (20-fold concentrate) was run on 7.5% SDS-polyacrylamide gels, and subjected to immunoblot analysis using commercially available goat anti-human ACE2 antibodies (1:500 dilution) (AF933, R&D Systems Inc., Minneapolis, MN, USA) as we previously described to characterize mouse shed ACE2 fragments by mass spectrometry (Xiao et al., 2014 (link)). Mouse kidney cortex lysates were used as controls (1.5–10 μg protein). Densitometric analysis of the protein bands was performed using Kodak ID image analysis software (Eastman Kodak, Rochester, NY, USA).
Twenty-five micro liter of concentrated media (20-fold concentrate) was run on 7.5% SDS-polyacrylamide gels, and subjected to immunoblot analysis using commercially available goat anti-human ACE2 antibodies (1:500 dilution) (AF933, R&D Systems Inc., Minneapolis, MN, USA) as we previously described to characterize mouse shed ACE2 fragments by mass spectrometry (Xiao et al., 2014 (link)). Mouse kidney cortex lysates were used as controls (1.5–10 μg protein). Densitometric analysis of the protein bands was performed using Kodak ID image analysis software (Eastman Kodak, Rochester, NY, USA).
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