Fig. 2. mTORC1 phosphorylates IRS-1 at S636/639 during 8-Br-cAMP-induced decidualization.

a Human endometrial stromal (hES) cells were induced to differentiate for 4 days in the presence of 0.5 mM 8-Br-cAMP. On day 0, 2, or 4 of differentiation, the cells were lysed and subjected to western blotting. b Cells were differentiated with or without 15 μM PF-4708671 at the initiation of differentiation for 2 days. Cells were lysed and then analyzed by either quantitative RT-PCR or western blotting. c On day 0, 2, or 4 of differentiation, the cells were lysed and subjected to western blotting. d Cells were differentiated for 2 days with or without 100 nM rapamycin at the initiation of differentiation for 2 days. e hES cells were infected with lentiviruses expressing two different raptor shRNAs or scrambled shRNA; cells were then selected by puromycin for 5 days. After 2 days of differentiation, the cells were lysed and then analyzed by either quantitative RT-PCR or western blotting. Human GAPDH was used to normalize gene expression. All blots are representative of three to five independent experiments. All data are shown as the mean ± SD, with paired t-tests performed as indicated. *P < 0.05