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. 2020 Nov 30;13(1):e12828. doi: 10.15252/emmm.202012828

Figure 2. Functional characterization of the decoy antibody.

Figure 2

  1. Competitive ELISA. ACE2‐Fc can compete with ACE2‐Fc‐Biotin for Spike 1‐674 binding in a dose‐dependent manner. The competition effects of ACE2‐Fc on ACE2‐Spike protein binding were normalized to PBS control. Error bars represent the standard deviation (SD), n = 2. Statistical analysis was performed by unpaired two‐tail t‐test. **P < 0.01, ***P < 0.001.
  2. Recognition of full‐length Spike on H1975 (left panel as negative control) or H1975‐Spike (right panel) cells by ACE2‐Fc using flow cytometry analysis. Isotype control: 40 μg/ml mouse IgG‐FITC.
  3. Confocal microscopy of H1975‐Spike‐overexpressing cells. Spikes on H1975 cells were stained with anti‐Spike antibody and Alexa Fluor® 594‐conjugated secondary antibody on ice for 1 h. After that, ACE2‐Fc‐FITC was incubated for another 1 h. Scale bars equal to 20 µm. DAPI was used as a nuclear counterstain.
  4. Preservation of ACE2‐Fc peptidase activity. The peptidase activity of ACE2‐Fc was measured by cleavage of the fluorescent peptide substrates.
  5. Inhibition of angiotensin II (Ang II)‐induced TNF‐α production by ACE2‐Fc. Ang II was preincubated with indicated amounts of ACE2‐Fc or IgG for 30 min. After that, the mixtures were added to RAW264.7 cells for 12 h. The concentration of TNF‐α in the culture medium was determined by ELISA. Error bars represent the standard deviation (SD), n = 3. Statistical analysis was performed by unpaired two‐tail t‐test. *P < 0.05. The dotted line represents the mean value of TNF‐α in control group.
  6. Inhibition of Ang II‐induced ADAM17 (a disintegrin and metalloprotease 17) phosphorylation by ACE2‐Fc. The protein extracts from (E) were immunoblotted with the indicated antibodies (left panel). β‐actin served as the loading control. The signal intensity was normalized to cells only (right panel). Data are representative of three independent experiments, and the values are expressed as the mean ± SD (right panel). Error bars represent the standard deviation (SD), n = 2. Statistical analysis was performed by unpaired two‐tail t‐test. *P < 0.05. IgG represents the human normal IgG control.