FIG 3.
Human (h), feline (f), and mouse (m) ACE2 orthologs are glycosylated, metabolically stable, and detected at the cell surface in stably transfected HRT-18G cells. (A) HRT-18G cells were stably transfected with IRES-Thy1.1 plasmids containing cDNAs of human (blue), feline (orange), and mouse (purple) ACE2 and magnetically sorted until equivalent levels of the reporter protein Thy1.1 were expressed on all three cell lines. Staining of parental HRT-18G cells is shown as a negative control (shaded histogram). (B) RNA was isolated from equal numbers of HRT-18G/hACE2, HRT-18G/fACE2, and HRT-18G/mACE2 cells. cDNAs were made from extracted RNAs of the tested cell lines, and qPCR was performed using GAPDH as a housekeeping gene. CT values from ACE2 amplifications were normalized to CT values from GAPDH, and the average fold changes from three independent experiments are shown. (C) To confirm the specificity of the ACE2 antibody for human ACE2, HRT-18G/hACE2 (blue), HRT-18G/fACE2 (orange), HRT-18G/mACE2 (purple), and HRT-18G (shaded histogram) cells were simultaneously incubated with fluorescently labeled ACE2 antibody and analyzed by flow cytometry. (D) HRT-18G cells expressing the indicated ACE2 orthologs were lysed and either mock treated or deglycosylated by incubation with PNGase F. Cell lysates were then resolved by SDS-PAGE and immunoblotted (IB) with polyclonal antibodies to ACE2 or β-actin. Molecular weight markers are indicated. (E) Cells were cultured with CHX for 0, 5, or 10 h, followed by lysis, SDS-PAGE, and Western blot analysis for either ACE2, β-actin, or LC3B. C, control: HRT-18G cells which do not express any ACE2 ortholog. (F) Cell surface proteins were biotinylated prior to cell lysis. Biotin-conjugated proteins were isolated using a streptavidin agarose slurry and analyzed by Western blotting for ACE2, E-cadherin, and β-actin.