Extended Data Fig. 3: Characterization of LCFA-CoA activation of AMPK.
From: Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK β1 isoforms
a, Activities of AMPKα1β1γ1 (COS7 cell-expressed, WT and β1S108A mutant) were determined by 32P SAMS assay, following immobilization on anti-myc agarose, ± palmitoyl-CoA, myristoyl-CoA or lauroyl-CoA (10 μM). Data are shown as mean fold change in AMPK activity vs. vehicle ± s.e.m., n=3. Statistical significance was calculated using two-way ANOVA with Bonferroni’s multiple comparisons test. n represent biological independent experiments. b, Activities of AMPKα1β1γ1 (Sf9 insect cell-expressed) were determined by TR-FRET in the presence of the indicated concentration of phosphatase PP2Cα ± AMP (30 µM) or palmitoyl-CoA (10 µM). Data are shown as mean fold change in AMPK activity vs. vehicle ± s.e.m., n=10 except for AMP incubated (n = 5). Statistical significance was calculated using two-way ANOVA with Bonferroni’s multiple comparisons test. n represent biological independent experiments. c, d, Binding of [3H]-palmitoyl-CoA to AMPKα1β1γ1 (E. coli-expressed) ± increasing concentrations of unlabeled palmitoyl-CoA (c), or to various AMPK preparations (d). GST-αRIM2: His6-GST-LVPRGS(thrombin cleavage site)-α1(282-374). Data are shown as mean relative binding ± s.e.m. For c, n = 2; for d, n = 3. Statistical significance was calculated using one-way ANOVA with Bonferroni’s multiple comparisons test. n represent biological independent experiments.
