Effect of Se-methylselenocysteine (MSC) on Akt phosphorylation. (a) Scheme for pretreatment of TM6 cells with MSC. Cells were synchronized in minimal medium for 24 hours. Cells were then exposed to 100 μM MSC for 3, 6, 10, 18 and 24 hours in minimal medium before being stimulated with fresh DMEM/F12 medium containing growth factors (5 ng/ml epidermal growth factor and 10 μg/ml insulin) and serum (2% adult bovine serum) for 1 hour. (b) Effect of pretreatment of MSC on Akt phosphorylation in TM6 cells. Equal amounts of lysates (30 μg of protein) were loaded on each lane for each time point. After electrophoresis the immunoblots were probed with anti-phospho-Akt (Ser473) antibody as described in the Materials and methods section, and the levels were measured with Molecular Dynamics software. Each bar represents levels in TM6 tumor cells treated with MSC in three different wells. *P < 0.05 compared with 0 hours.