Cell surface biotinylation was performed in a similar manner. However, pEGFP-AQP2-F204V, was transfected into MDCK cells stably expressing wild-type AQP2 and cells made stable with vector alone. Twenty-four hours post-transfection, cells were stimulated with forskolin, trypsinized, resuspended in 1 ml of PBS (2.5 × 106 cells/ml), and incubated with 0.5 mg of NHS-PEO4-biotin (Pierce Biotechnology) for 30 min at room temperature. Cells were washed once in 10 mM Tris (pH 8) and three times in PBS, after which membranes were purified and solubilized as described above. Solubilized membranes were incubated with 20 μl of immobilized streptavidin (Pierce Biotechnology) for 2 h at 4 °C. Finally the precipitated proteins were washed in RIPA buffer and boiled in 50 μl of Laemmli buffer. Total cells and the biotinylated precipitates were immunoblotting using an antibody to AQP2.