Mab933

In order to assess overall protein localization of ACE2 variants, A549 cells grown at 40000 cells/well in 8-well cell culture chamber (Sarstedt, #94.6170.802) were transfected with 1,5 μL Lipofectamine 3000^® (Thermo Scientific, #L3000015) following the manufacturer’s protocol. At 48h post transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature (RT) and washed twice with PBS. Then, they were permeabilized with 0.1% Triton X-100- PBS for 15 min and incubated with 2% BSA in PBST (PBS+ 0.1% Tween 20) for 1 h at RT to block nonspecific binding. Chambers were incubated with either anti-ACE2 (1:100; MA5-32307; Thermo Scientific) or anti-ACE2 (1:50; MAB933; R&D Systems) in PBST containing 0,1% BSA ON at 4°C, washed twice in PBS and incubated with Alexa-Fluor647-labeled anti-rabbit (1:2000; ab150079; Abcam) or anti-mouse (1:2000; ab150115, Abcam) for 1h at RT. Images were acquired with Cell Observer-Zeiss.

Total proteins from EndoC-βH1 cells were extracted using a lysis buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 1% (w/v) NP-40, 2 mM EDTA) supplemented with 1× protease inhibitors (Roche). Total proteins were quantified using Bradford assay, and 50–100 μg protein/lane was separated using SDS-PAGE Tris-Glycine gradient Bis-Acrylamide gel 4–20%. Proteins were then transferred to Nitrocellulose 0.2 μm membrane using wet electrophoresis system. Upon transfer onto nitrocellulose, membranes were washed three times with TBST 1× (Tris-HCl 25 mM, NaCl 150 mM, Tween 20 0.1%, pH 7.4) and then incubated 2 h with 5% non-fat dry milk in TBST 1×. To identify ACE2, three different antibodies were used: #Ab108252, #Ab15348 (Abcam), and #MAB933 (R&D system) were respectively diluted 1:1,000, 1:500, and 1:250 in 5% non-fat dry milk in TBST 1× and incubated o/n at +4°C and then with Goat anti-rabbit (#111-036-003, Jackson Laboratories) or Goat anti-Mouse (#115-036-003, Jackson Laboratories) diluted 1:5,000 in 2% non-fat dry milk in TBST 1× 1 h RT. After three washes with TBST 1× and one wash in TBS 1×, chemiluminescent signal was detected by using ECL solution (GE Healthcare, Little Chalfont, Buckinghamshire, UK-RPN2232). Chemiluminescent analysis of immunoblot results was performed by using LAS400 analyzer (GE Healthcare, Little Chalfont, Buckinghamshire, UK-RPN2232).