Kidney membrane fractions (60 μg) were resolved on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.05% Tween 20 (TBST), followed by an overnight incubation (at 4 °C) with AQP2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States; #sc-9882). Membranes were washed in TBST then incubated with HRP-conjugated donkey anti-goat antibody. Membranes were washed further in TBST and bands were visualized using ECL reagent (Amersham Biosciences, Little Chalfont, United Kingdom).