Assessment of cell surface expression levels of human ACE2

Quantification of ectopic hACE2 expression on the surface of HEK293 cells was achieved using Pierce Cell Surface Biotinylation and Isolation kits (Thermo Fisher), according to the manufacturer’s instructions. Briefly, HEK293T cells cultured in 10 cm dishes were transfected with equal amounts of pcXN2 plasmid expressing full-length hACE2 wt or glycan deletants. At 24 h post transfection, cells in each dish were washed with 4 ml of ambient temperature PBS and biotinylated using 5 ml of Sulfo-NHS-SS-Biotin Solution for 10 min at RT. After removing the labeling solution, the biotin-labeled cells were washed twice with 10 ml ice-cold TBS. The cells were then scraped off, transferred into 1.5 ml micro-tubes and lysed with 180 μl of Lysis Buffer for 30 min at RT. The lysate was clarified by centrifugation at 15,000 rpm for 5 min at 4 °C to remove the cell debris (Input samples). To normalize the amount of Input samples, the protein concentration of clarified cell lysate from each dish was measured using Pierce BCA Protein Assay Kits (Thermo Fisher). An equal amount of Input samples was mixed with 250 μl slurry of NeutrAvidin resin for 30 min at RT, washed with Wash Buffer and then eluted with 200 μl of DTT solution. Extracellular levels of hACE2 wt and glycan deletants on the cells surface in Input samples were analyzed by western blotting as described below.