HEK 293 cell lysates (40 ug) or immunoprecipitates undertaken from 2 mg of HEK 293 cellular lysates with anti-NKCC1 (1-260) (5 ug) or pre-immune IgG were subjected to SDS-Page, transferred to nitrocellulose membrane and immunoblotted with anti-NKCC1 (1-260) (2 ug/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).