Representative Western blots of angiotensin I-converting enzyme (ACE) protein detection.
Western blots shown in the plasma (A), homogenized lung (A,B), kidney (B) and testis (b) from naive LEW/N and F344/N rats. Purified human ACE, used as a standard, and various amounts of total protein from plasma and homogenized tissues (20 µg for plasma and kidney, 5 µg for testis, 2 to 3 µg for lung) were run by SDS-PAGE on a NuPAGE tris-acetate 3–8% gel. Proteins were transferred to the PVDF membrane and incubated over night at 4°C in the blocking buffer with the rabbit polyclonal anti-ACE antibody (1:4000). The detection was performed using an anti-rabbit (IgG-HRP) (1:10000) and an enhanced chemiluminescent plus (ECL +) detection system. Blot was scanned in blue chemifluorescence mode (100 µ pixel size, PMT 1000 V) by the Storm 860. (C) Representative histogram of ACE quantification in homogenized tissues. The ACE bands were quantified using ImageQuant software. The quantification was performed using the linear portion of the standard curve made by serial dilution of pure ACE. Data are expressed as ng of ACE per µg of total protein.