Fig. 3: AMPK phosphosites sensitive to pharmacological inhibition of mTORC1.

A Sequence alignment of selected Ser-Pro phosphosites on AMPK with the mTOR consensus motif²³. B HEK293T/17 cells expressing α1β1γ2 or α2β2γ2 were treated with rapamycin (100 nM) or torin1 (1 µM) for up to 4 h and lysates immunoblotted as indicated. Representative immunoblots from 3 independent experiments are shown. C α1β1γ2 and (D) α2β2γ2 complexes from (B) were FLAG-immunoprecipitated and subjected to tryptic digest. Peptides and phosphopeptides were detected by LC-MS and area under the curve was used to calculate stoichiometries; rapamycin and/or torin1 sensitive phosphosites at 1 h and 4 h post-treatment are depicted here. Data presented as mean % stoichiometry ± SEM, n = 3. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. basal. n.s. not significant.