Figure S2.

Characterization of Binding between SARS-CoV-2 and hACE2 by Flow Cytometry, Related to Figures 1 and 2

(A-C) Supernatant containing the indicated mFc-fusion proteins were incubated with HEK293T cells transiently expressing eGFP-tagged hACE2 (A), hCD26 (B) or hAPN (C), respectively. Anti-mIgG/APC was used to detect the mFc-fusion protein binding to the cells. Culture supernatant of HEK239T cells was used as negative control and marked as NC. For each sample, eGFP positive cells were first gated and then used to analyze fluorescence intensity of APC.

(D-F) Supernatant containing SARS-CoV-2-CTD-mFc proteins were pre-incubated with soluble hACE2 (D), hCD26 (E) or hAPC (F) at the indicated concentrations before addition to HEK293T cells transfected with pEGFP-N1-hACE2. mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC.

(G-I) Supernatant containing SARS-RBD-mFc proteins were pre-incubated with soluble hACE2 (G), hCD26 (H) or hAPC (I) at the indicated concentrations before addition to HEK293T cells transfected with pEGFP-N1-hACE2. mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC.

(J-K) HEK293T cells transfected with pEGFP-N1-hACE2 were pre-incubated with soluble SARS-RBD at the indicated concentration, before the addition of supernatant containing either SARS-CoV-2-CTD-mFc (J) or SARS-RBD-mFc (K). mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC.

(L-M) HEK293T cells transfected with pEGFP-N1-hACE2 (WT), or the mutants containing K353A (K353A) or K31A (K31A) were incubated with supernatant containing either SARS-CoV-2-CTD-mFc (L) or SARS-RBD-mFc (M). mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC.

All data shown are representative of two independent experiments.

The fluorescence signals were monitored by BD FACSCanto and the results were analyzed using FlowJo V10 (https://www.flowjo.com/solutions/flowjo/downloads).