Figure 1.

A. ACE2 activity in transfected Neuro-2A cells in control (white bar), or after treatment with Ang-II alone (100 nM, black bars), or Ang-II and losartan (1 μM, horizontal hatched bars), or Ang-II and leupeptin (100 μM, vertical hatched bars) after 4 h (middle columns) or 18 h (right columns). ACE2 activity was determined as described in Experimental Procedures and the results were expressed as % from the activity measured in control cells (100% corresponds to 449 ±51 FU/min/μg of proteins). n=12 from 5 independent transfections, *−indicates P<0.05. B. Plasma membrane levels of ACE2 in control conditions and after treatment with 100 nM Ang-II for 4 and 18 h. Neuro-2A cells were transfected with 2 μg/well ACE2-GFP in 6-well plates and 48 h later the plasma membrane proteins were isolated by biotinylation, as described in Methods section. Subsequently, ACE2 levels were determined by western blot using ACE2 (top) or GFP (middle) antibodies. Na⁺/K⁺ ATP-ase (bottom) was used as a loading control. The summary data of three independent experiments are shown in the bottom panel. *−indicates P<0.05. C. Total cellular levels of ACE2-GFP in Neuro-2A cells in non-treated controls and after Ang-II (100 nM) treatment for 18 h. Similar results were obtained in two other independent experiments. FU: Fluorescence units.