Effect of Se-methylselenocysteine (MSC) on phospho-Akt, phospho-Raf and phospho-MEK in TM6 cells. The TM6 cells were synchronized in minimal medium as described in Fig. 1, but only for 24 hours. Then the MSC (100 to 400 μM) was added (in minimal medium) for 16 and 24 hours. The cells were stimulated with fresh DMEM/F12 containing growth factors (5 ng/ml epidermal growth factor and 10 μg/ml insulin) and serum (2% adult bovine serum) at the indicated time points for 1 hour. Equal amounts of lysates (20 μg of protein) were loaded on each lane for each time point. Immunoblots were probed with anti-phospho-Akt, anti-phospho-Raf, anti-phospho-MEK and anti-actin antibodies as described in the Materials and methods section. Both the Akt and Raf were phosphorylated within 1 hour of stimulation with growth factor and serum. At 16 hours a dose of 400 μM MSC failed to inhibit the phosphorylation of Akt or Raf and the downstream effector MEK. However, at 24 hours MSC was able to inhibit phosphorylation of all three proteins in a dose-dependent manner.