Fig. 5. Determination of cell-surface AQP2 immunoreactivity. The anti-AQP2 C-loop antibody was employed to monitor the AQP2 density on the plasma membrane in CD8 cells. (A) In non-permeabilized cells, the antibody is expected to cross-react only with AQP2 inserted into the plasma membrane. (B) After stimulation of untreated cells with forskolin, the immunodetectable AQP2 on the cell surface increased by approximately two-fold compared with that present in the plasma membrane in control cells (-TeNT). By contrast, TeNT pretreatment completely abolished forskolin-stimulated AQP2 targeting to the apical plasma membrane, as assessed by quantification of cell-surface immunoreactivity (Fig. 6B, +TeNT). The results shown represent the means±s.e.m. of three separate experiments in which about 4x10⁶ cells (six separate wells) were tested for each experimental condition in each experiment. (C) Double labeling of AQP2 and VAMP-2 in cells transiently transfected with GFP-tagged AQP2 showed a partial colocalization of VAMP-2 in AQP2 bearing vesicles. Bar, 5 µm.