Effect of estrogen treatment on p-S2448-mTOR and p-p70S6Kinase in MCF7 human breast cancer cells. MCF7 were serum starved and estrogen depleted and then treated with and without E2 (10 nM) for the indicated time periods. Cell extracts were prepared and analyzed as described in the Methods section: 100 ug of extr0061ct was subject to Western blotting for p-mTOR, total mTOR, p-p70S6K and total p70S6K. Results represent one of four to seven independent experiments. Upper panel: for p-S2448-mTOR, E2 treatment for three hours showed a 1.5 ± 0.2 fold, n = 7 (mean ± SD) and at six hours 1.4 ± 0.3 fold, n = 5 (one way ANOVA P = 0.0006) increase. Middle panel: for p-T389-p70S6K, E2 treatment for three hours showed a 1.9 ± 0.5 fold, n = 10 (mean ± SD) and at six hours 1.6 ± 0.3 fold, n = 10 (one way ANOVA P < 0.0001) increase. ANOVA, analysis of variance; E2, estradiol; mTOR, mechanistic target of rapamycin; p-S2448 mTOR, mTOR phosphorylated on serine 2448; SD, standard deviation.