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. 2004 Dec 1;18(23):2893–2904. doi: 10.1101/gad.1256804

Figure 4.

Figure 4.

mTOR regulation by hypoxia is both AMPK and Lkb1 independent. (A) Western blot analysis of extracts of Tsc2+/+ and Tsc2-/- MEFs treated with AICAR for the indicated periods of time. (ACC-P) Acetyl-CoA carboxylase phosphorylated at S79; (AMPK-P) AMPK phosphorylated at T172. (B) Western blot analysis of Tsc2+/+ MEFs after 4 h of treatment with either hypoxia or AICAR. (C) In vitro AMPK kinase assay of the same extracts used in B immunoprecipitated in antibody excess with a polyclonal anti-AMPK (α AMPK) antibody or normal rabbit IgG (IgG). Samples were normalized for protein concentration prior to immunoprecipitation. Error bars equal one standard deviation (n = 3). Also shown are background activities of immunoprecipitates incubated in the absence of substrate (SAMS peptide). (D) Western blot analysis of Tsc2+/+ cells pretreated or not with the AMPK inhibitor compound C and exposed to either AICAR or hypoxia for the indicated periods of time. All cells treated with compound C were exposed to the drug for 9.5 h. (E,F) Western blot of Lkb1+/+ or Lkb1-/- MEFs treated with AICAR (E) or hypoxia (F) for the indicated periods of time.