Anti ace2

Cell were lyzed with RIPA buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5) and lysates were incubated with either anti-ACE2 or anti-vimentin antibody-conjugated protein G agarose beads (Abcam) overnight at 4°C. Beads were then washed with RIPA 1X buffer and bound proteins were eluted with Glycine 0.2M, pH 2.5. Eluted proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by Western blot. Membranes were saturated using PBS with 0.05% Tween 20 (PBST) supplemented with 3% powdered milk for 45 min and incubated with anti-vimentin (Santa Cruz) or anti-ACE2 (Santa Cruz) antibodies overnight at 4°C on a shaker. Membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce) for 2 h. Proteins were visualized using the chemiluminescence reaction HRP Substrate (Millipore), and image acquisition was done by using a Fusion Fx imaging system (Vilber Lourmat).
For detection of vimentin secretion, cell supernatants were centrifuged or not to remove cell debris, subjected to lyophilization for 24 h and resuspended in RIPA buffer. Culture medium alone was processed accordingly as negative control. Proteins were then separated by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by Western blot.