FIGURE 1.

Rapamycin inhibits the phosphorylation of GSK3-β (S9) in LPS-stimulated cells. A, Human monocytes were stimulated with LPS (1 μg/ml) in the presence or absence of rapamycin (100 ng/ml) for 60 min, and the levels of phosphorylated GSK3-β (S9) and total GSK3-β were analyzed using a phospho-Ab array. B, The mean ratio of GSK3-β (S9) to total GSK3-β is shown ± SD of six experiments. C, Wild-type and Rictor-deficient MEFs were treated with LPS (1 μg/ml) over a 2-h time course, and the levels of phosphorylated GSK3-β (S9) were analyzed by Western blot. D, Densitometry scans were performed and the mean ratios of phosphorylated and total GSK3-β are shown ± SD of three experiments. E, Human monocytes were stimulated with LPS (1 μg/ml) in the presence or absence of rapamycin (100 ng/ml) for up to 24 h, and the levels of phosphorylated GSK3-β (S9) were analyzed by Western blot. F, Densitometry scans were performed, and the mean ratios of phosphorylated and total GSK3-β are shown ± SD of three experiments. G, The levels of phosphorylated GSK3-β (S9) in LPS-stimulated human monocytes treated with or without rapamycin (100 ng/ml) were monitored by flow cytometry. H, The effects of rapamycin (100 ng/ml) on the levels of phosphorylated GSK3-β (S9) and the GSK3-specific substrate, glycogen synthase (S641), in LPS-stimulated cells were monitored by Western blot. I, Inhibition of mTORC1 using rapamycin (100 ng/ml) and its effects on the levels of phosphorylated mTOR, p70S6K, p85S6K, and 4E-BP1 in LPS-stimulated monocytes. J, Inhibition of GSK3 using SB216763 (10 μM) and its effects on the phosphorylated levels of mTOR, p85S6K, and p70S6K in LPS-stimulated monocytes. Data are representative of three to six separate experiments. **p < 0.01, statistically significant differences between monocytes stimulated with LPS in the presence or absence of rapamycin.