2.7. Co-Immunoprecipitation of mTOR and In Vitro mTOR Kinase Assay

Rh30 cells were seeded in 100 mm culture dishes (3 × 10⁶ cells/dish) and grown overnight. The cells were then treated with DHA (0–30 μM) for 24 h. After aspirating the used medium, the cells were briefly washed with PBS and lysed in an ice-cold CHAPS lysis buffer, followed by immunoprecipitation with goat anti-mTOR antibody or normal goat IgG (as a control). Finally, to detect the interaction of mTOR with raptor, rictor, and mLST8, the immunoprecipitants were subjected to immunoblotting with antibodies to mTOR, raptor, rictor, and mLST8, as described [41]. To detect the mTORC1 activity, the above immunoprecipitants were utilized for the in vitro mTOR kinase assay, as described [41].