Fig. 3

Overexpressed WT ACE2 subcellular localization and stability. A Immunofluorescence confocal imaging of permeabilized HeLa cells transfected with Flag-tagged ACE2 (red), GFP-tagged HRas (plasma membrane marker in green) and Calnexin (ER marker in blue). Images were acquired using 100X magnification and manipulated by ImageJ. Scale bar = 50 μm. B HEK293 cells transiently transfected with GFP and Flag-tagged WT ACE2 plasmids for 48  Hrs. Lysates were then digested in presence or absence of Endo H and PNGase F enzymes for 3 Hrs and 1  Hrs, respectively. Anti-Flag primary antibody was used to stain WT ACE2 and anti-GFP antibody was used to satin GFP that was used as a transfection control. C Flag-tagged WT ACE2 transfected cells were treated with DMSO and 100 μg/ml cycloheximide for a period of 18 Hrs. Cells were lysed at the indicated time points (0, 4, 8, 12, and 18 Hrs) and lysates were analyzed by western blotting. Anti-Flag antibody was used to stain WT ACE2. Mock sample represent un-transfected cells. In all the experiments actin was used as a loading control. D Graph representing the relative expression of WT ACE2 compared to DMSO treated cells at the indicated time points. Error bars represent ± SEM of three independent experiments. Band quantification was performed using ImageJ