Rictor was immunoprecipitated from either HEK 293 cellular extracts using 2 ug of anti- Rictor per amount of extract indicated. Immunoprecipitates were subjected to SDS-Page analysis before being transferred to nitrocellulose membrane and immunoblotted with anti-Rictor 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).