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. 2009 Dec 18;285(11):7866–7879. doi: 10.1074/jbc.M109.096222

FIGURE 5.

FIGURE 5.

Energy stress down-regulates mTORC1-associated mTOR Ser-2481 autophosphorylation. A, energy stress mediates mTORC1-associated mTOR Ser-2481 dephosphorylation. Cycling HEK293 cells were untreated (lanes 1 and 2) or treated with 2DG) (25 mm) for 15 min (lanes 3–6). After 2DG treatment, cells were re-fed with media lacking 2DG and incubated for an additional 30 min (lanes 5 and 6). WCL was immunoprecipitated with anti-raptor antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expected activation of AMPK and inhibition of mTORC1 signaling by 2DG. B, in MEFs, energy stress and amino acid withdrawal mediates mTORC1-associated mTOR Ser-2481 dephosphorylation. Cycling wild type MEFs (lanes 1 and 2) were incubated in D-PBS/Glc/FBS for 60 min to effect amino acid (AA) deprivation (lanes 3 and 4) or treated with 2DG (25 mm) for 15 min (lanes 5 and 6). WCL was immunoprecipitated with anti-raptor antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expected activation and/or inhibition of mTORC1 signaling by the various treatments.