Effect of lysomotropic agents on the cell-surface expression and biosynthesis of ACE2. (A) Vero E6 cells were cultured for 20 h in the absence (control) or presence of chloroquine (10 μM) or NH4Cl (20 mM). Cells were labeled with anti-ACE2 (grey histogram) or with a secondary antibody alone (white histogram). (B) Biosynthesis of ACE2 in untreated cells or in cells treated with NH4Cl. Vero E6 cells were pulse-labeled for 3 h with 35S-Met, and the cell lysates were immunoprecipitated with an ACE2 antibody (lane 1). Preincunbation of the antibody with recombinant human ACE2 (rhACE2) completely abolished the signal (lane 2). The positions of the endoglycosidase H-sensitive ER form and the endoglycosidase H-resistant Golgi form of ACE2 are emphasized. Note that the increasing concentration of NH4Cl resulting in the decrease of the Golgi form of ACE2. (C) A similar experiment was performed in the presence of the indicated concentrations of chloroquine. Note the loss of terminal glycans with increasing concentrations of chloroquine. (D) The terminal glycosidic modification of ACE2 was evaluated by neuraminidase treatment of immunoprecipitated ACE2. Here cells were treated with 1–25 μM concentrations of chloroquine during starvation, pulse, and 3-h chase.