Fig. 7: dACE2 is non-functional for binding SARS-CoV-2 spike protein RBD and as a carboxypeptidase.

a, Representative confocal images of T24 cells transiently overexpressing dACE2–Myc (white) or ACE2–GFP (green) and treated with the RBD of the SARS-CoV-2 spike protein (red); nuclei (DAPI), blue. Scale bars, 20 µm. b–e, Representative flow cytometry histograms (b,d), and mean fluorescence intensity (MFI) values from three biological replicates (c,e), of spike RBD binding to the surface of ACE2–GFP- but not dACE2–Myc-expressing T24 cells. Gating for cells expressing dACE2–Myc, ACE2–GFP or both proteins is shown in Extended Data Fig. 9a. The results are based on three biological replicates, and represent one of two independent experiments. f, SARS-CoV-2 infectivity rates (%) in a lung cancer cell line, A549, transfected with GFP or dACE2–Myc, or stably expressing ACE2 (ACE2-stable) and transfected with GFP. g, The SARS-CoV-2 viral load as ddCt values compared to mock, corresponding to the plot in f. Additional details are provided in Extended Data Fig. 10. h, A representative western blot with an anti-ACE2 antibody that detects both recombinant ACE2–GFP and dACE2–GFP overexpressed in T24 cells. The amount of the ACE2–GFP lysate was kept constant, while the amount of the dACE2–GFP cell lysate was increased and the difference in the lysate volume was compensated by the empty GFP vector. i, The results of carboxypeptidase assays using variable amounts of lysates of cells (as described in the plot in h), showing that the activity of ACE2 is not affected by increasing amounts of dACE2. The results are based on three biological replicates and are presented with means and s.d. The western blot shows the results of one representative replicate.