Fig. 3
From: Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism
Deciphering allosteric activation and protection against dephosphorylation. a FRET ratios of AMPfret 1.0 wild type (WT) and corresponding CBS site mutants in presence of AMP (30 μM, orange bars) or ADP (200 μM, yellow bars) were normalized to the adenylate-free control (blue bars). Data and error bars represent mean ± SEM (n ≥ 5). b AMP-induced allosteric activation of AMPfret WT and its CBS site mutants. AMPK activity was quantified with increasing concentrations of AMP and fixed MgATP (200 µM) by immunoblotting for phosphorylated acetyl-CoA carboxylase (P-ACC; see Supplementary Fig. 5) and data normalized to kinase activity in absence of AMP. Data and error bars represent mean ± SEM (n = 3). c, d AMP- and ADP-dependent protection against dephosphorylation of AMPfret WT and its CBS site mutants. AMPK activity was quantified with pre-phosphorylated AMPK, phosphatase PP2Cα and increasing concentrations of AMP or ADP by immunoblotting for α-subunit P-T172 (see Supplementary Fig. 5) and data normalized to dephosphorylation in absence of ADP. Data and error bars represent mean ± SEM (n = 3). After checking normality and equality of variance, statistical significance was analysed by two-way ANOVA followed by Student–Newman–Keuls multiple comparison. Means sharing the same letter do not differ significantly (p ≤ 0.02)
