Fig. 5. No effect of anti-hACE2 mAbs on hACE2 activity and distribution.

a,b, The ACE2 inhibitor control MLN-4670 (a) or anti-hACE2 mAbs (2G7A1, 05B04, 05H02 and 05B04LC/05D06HC, 2 μg ml⁻¹, 10 μg ml⁻¹ and 50 μg ml⁻¹) (b) were mixed with hACE2 (0.2 μg ml⁻¹) and a fluorogenic ACE2 substrate in 96-well plates. After incubation, fluorescence intensity (555 nm/585 nm, excitation/emission), indicative of hACE2 enzymatic activity, was measured and plotted as a percentage of the uninhibited control. Mean and range of four independent experiments is plotted. c, Outline of an assay to determine hACE2 and anti-hACE2 mAb internalization. Live A549 cells expressing a C-terminally (intracellular) HA-tagged hACE2 receptor were incubated with anti-hACE2 mAbs, Then, cells were fixed and permeabilized. Total hACE2-HA was then immunostained with a mouse anti-HA-tag antibody. The internalization of hACE2-HA and anti-hACE2 mAbs was then evaluated by staining with anti-mouse Alexa Fluor 594 (Alexa-594) and goat anti-human Alexa Fluor 488 (Alexa-488), respectively. d, Localization of anti-hACE2 mAbs (green, left) or hACE2-HA (red, centre) or both (right) following incubation of live A549/hACE2-HA cells with 2G7A1, 05B04 or 05H02, or the absence of human anti-hACE2 antibody as indicated. Blue stain (DAPI) indicates cell nuclei. Scale bars, 10 μm. Representative of three independent experiments.

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