Fig. 5: Longer exposure of HEK293T cells to torin1 leads to gradual loss of β-pS182/184 underpinned by slow in vitro dephosphorylation kinetics.

HEK293T cells expressing FLAG-fusions of (A) α1β2γ1, (B) α2β2γ1, (C) α1β1γ1, or (D) α2β1γ1 were incubated with 250 nM torin1 for up to 24 h, and indicated phosphosites/stoichiometries detected from prepared lysates (WCL) or FLAG-immunoprecipitated AMPK by immunoblot or LC-MS. Data presented as mean phosphorylation (fold vs. baseline (0 h)) ± SEM, or 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 and ****P < 0.0001 vs. basal. E Lysates were prepared from HEK293T cells incubated with rapamycin or torin1 (0.05 to 1 μM) for 24 h and endogenous immunoblotted as indicated, with quantitation presented as mean phosphorylation (fold vs. basal) ± SEM, n = 4. F iRAPWT or iRAPKO MEFs were incubated with 1 μM 4-OHT for 96 h and prepared lysates immunoblotted as indicated. Data presented as mean phosphorylation (a.u. arbitrary units) ± SEM, n = 3. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple-comparisons test. n.s. not significant vs. vehicle untreated. COS7 cells expressing GST fusions (GST-α1) of (G) α1β1γ1 or (H) α1β2γ1 were incubated with phenformin (2 mM, 1 h), H₂O₂ (5 mM, 45 min) or 2-deoxy-glucose (2DG; 25 mM, 30 min), and GST-purified AMPK was immunoblotted as indicated. I Lysates were prepared from immortalised MEFs (WT or SIN1^−/−), incubated under conditions of serum starvation overnight ± subsequent serum re-addition (1 h), and immunoblotted as indicated. Representative immunoblots from three independent experiments are shown.