Nedd 4.2 was immunoprecipitated from 0.2 mg of clarified PC12 cellular extract using 0.5 μg of anti-Nedd 4.2 antibody. The immunopecipitate was resolved by SDS-Page on a 4-12 % Bis-Tris gel and transferred to PVDF membrane. The membrane was then probed with anti-Nedd 4.2 antibodies 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).