Research Paper Volume 2, Issue 8 pp 487—503

Figure 3. (A) Phospho-specific immunoblot analysis of mTOR/S6 kinase cascade activity under different cell feeding conditions. Cells were incubated for 24 hours in the indicated conditions (ctrl= 2g/l glucose + Aminoacids; noAA= glutamine and NEAA omitted). Where possible the same filter was cut into parallel strips and hybridized contemporarily with different antisera. When molecular weights of target proteins overlapped, filter were stripped and re-hybridized, or twin filters were prepared with the same protein lysates. Hyperphosphorylated protein species usually migrate slower and are indicated by separate arrows. Picture representative of several independent experiments. (B) Effect of metabolic inhibitors from figure 1B on S6 kinase phosphorylation. Upper arrows indicate the fully phosphorylated forms. Equal content of total S6 kinase in the different samples was verified by anti total S6K immunoblotting of the same protein lysates on a different nitrocellulose membrane. Picture representative of 2-3 three independent experiments.