Af3437

To generate short ACE2 fragments that exhibit ACE2 activity, a series of ace2 DNA constructs of varying lengths were generated through truncation from the C terminus (Supplementary Materials Figure S1). The cDNA of short ace2 was generated by PCR amplification using as a template the cDNA of the intact soluble mouse ace2 (coding amino acids 1-740). To gradually shorten ACE2, we used specific primers that determine the length of the shorter ACE2 cDNA to be amplified and have compatibility with the expression vector restriction sites (pcDNA, Invitrogen, Carlsbad, CA, USA). The absence of mutations in the amplified cDNA was verified by sequencing. The plasmids with the inserted cDNAs of the short ACE2 variants were then expressed by transient transfection in HEK293 cells. ACE2 activity was measured in the conditioned culture medium using the fluorometric substrate Mca-APK-Dnp [7 (link)] (Supplementary Materials Figure S1). The native rACE2 that contained the full extracellular domain (1-740 AA) was used as a positive control. As a negative control, conditioned media from mock-transfected cells was used. In addition, Western blot using a polyclonal antibody raised against the entire extracellular domain of ACE2 (R&D Systems, AF3437) was used to detect the transgenes and confirm the molecular size of the over-expressed small ACE2 variants.

The antibodies used were as follows: Anti-rabbit antibodies against TGF-β1 (3711), total Smad-2/3 (3102), phosphorylated Smad-2/3 (3101), Smad-4 (9515), Smad-6 (9519), Bax (2772), Bcl-2 (2870), total caspase-3 (9662), cleaved caspase-3 (9661), cyclin B1 (4138), phosphorylated cdc2 (Tyr 15) (9111), phosphorylated histone H3 (Ser 10) (3377; Cell Signaling Technology, Beverly, MA), AT₁R (SC-1173; Santa Cruz Biotechnology, Santa Cruz, CA) and Mas receptor (AAR-013; Alomone Laboratories, Ltd., Jerusalem, Israel); anti-goat antibodies against connective tissue growth factor (CTGF) (SC-14939), ACE (SC-12187) and tumor necrosis factor-α converting enzyme (TACE) (SC-6416; Santa Cruz Biotechnology); anti-mouse antibodies against ACE2 (AF-3437; R&D systems, Minneapolis, MN), Ang II (NB100-62346; Novus Biochemicals, Littleton, CO), fibronectin (SC-71114; Santa Cruz Biotechnology, Santa Cruz, CA), α-smooth muscle actin (SMA) (A2547) and β-actin (A3854; Sigma) were commercially obtained.