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. 2009 Jan 28;29(4):1093–1104. doi: 10.1523/JNEUROSCI.4103-08.2009

Figure 1.

Figure 1.

EGF stimulation of astrocytes from adult spinal cord activates the mTOR pathway. A, EGF promotes phosphorylation of the EGF receptor and S6 kinase. Astrocytes were stimulated for 15 min with EGF (+) or vehicle (−), and lysates were probed by immunoblotting with antibodies to phosphorylated EGF receptor (p-EGFR) and phosphorylated S6 kinase (p-S6K) and reprobed with antibodies to the EGF receptor and S6 kinase. B, The Erk MAP kinase pathway is not involved in EGF-dependent S6 kinase phosphorylation. Astrocytes treated with the PD98059 inhibitor (+) or vehicle (−) were stimulated with EGF, and lysates were probed by immunoblotting with antibodies to phosphorylated S6 kinase (p-S6K) or phosphorylated Erk1 and Erk2 MAP kinases (p-Erk) and reprobed with antibodies detecting the nonphosphorylated forms of the proteins. The levels of phosphorylated S6 kinase determined from the optical density of the bands were normalized to total S6 kinase protein levels. The histogram shows average levels of phosphorylated S6 kinase relative to the level after EGF treatment. The error bars represent the SE from three independent experiments. PD98059 did not cause significant differences in S6 phosphorylation as determined by one-way ANOVA and Bonferroni's post hoc test. C, The PI3 kinase–Akt pathway mediates EGF-dependent Tuberin phosphorylation. Astrocytes incubated with the LY294002 PI3 kinase inhibitor (+) or vehicle (−) were stimulated with EGF, and lysates were probed by immunoblotting with the indicated antibodies. The levels of phosphorylated proteins were quantified as described in B for S6 kinase. The histogram shows average levels of phosphorylated Tuberin and phosphorylated Akt relative to the levels after EGF treatment. The error bars represent the SE from three independent experiments. ***p < 0.001 for the comparison between untreated and LY294002-treated by one-way ANOVA and Bonferroni's post hoc test. D, The mTORC1-selective inhibitor rapamycin blocks S6 kinase phosphorylation but not Akt phosphorylation. Astrocytes incubated with rapamycin (+) or vehicle (−) were stimulated with EGF, and the lysates were probed by immunoblotting with the indicated antibodies.