Rat tissue extracts (40 μg of protein) and HEK 293 cellular extract (40 μg of protein) were subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-WNK1 at 1 μg/ml. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).