Anti ace2 antibody clone 460502

For Western blotting, lung tissues were obtained 24 h after intratracheal instillation of the vehicle control or H1N1 and H5N1 viruses (10⁶ TCID₅₀). Tissues were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.0% Triton X-100, 20 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitors), the tissue lysates separated by SDS-PAGE, and the proteins transferred onto a nitrocellulose membrane. The membranes were incubated with antibodies against ACE, ACE2, and β-actin, followed by incubation with horseradish peroxidase–conjugated secondary antibodies. The anti-ACE antibody (2E2, sc-23908, 1:1,000) was purchased from Santa Cruz Biotechnology, Inc. The anti-ACE2 antibody (clone #460502, 1:300) was from R&D systems. The anti-β-actin antibody (clone AC-15, A5441, 1:10,000) was purchased from Sigma-Aldrich. The anti-Flag-Tag mouse mAb (LK-ab002-100, 1:5,000) was from Multisciences for validation the plasmid transfection efficiency in 293T cell line. The anti-ACE2 antibody (K465, 1:500) was purchased from Bioworld. Horseradish peroxidase (HRP)-conjugated secondary antibodies and Western Blotting Luminol Reagent were from Santa Cruz Biotechnology. Antibody binding was visualized using Kodak detection system and analyzed using Quantity One software.