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. 2013 Dec 19;4:3017. doi: 10.1038/ncomms4017

Figure 1. Regulation of AMPK in vitro and in HEK293 cells.

Figure 1

(a) Structures of A-769662 and 991 compounds, the rings of 991 are numbered. (b) 991 (red) allosterically activates recombinant α2β1γ1 with half maximal activation (A0.5) of 0.09±0.02 μM compared to 0.39±0.03 μM for A-769662 (black). Results are the mean±s.e.m. from at least three independent experiments. Inset, 991 protects against pT172 dephosphorylation of α1β1γ1 (grey) and α2β1γ1 (white) complexes. (c) Top, 991 (grey bars) activates endogenous AMPK in HEK293 cells at lower doses than A-769662 (white). HEK293 cells were treated with varying concentrations of A-769662 or 991, and endogenous AMPK was immunoprecipitated from cell lysates using a pan-β-specific antibody. AMPK activity was measured using the SAMS peptide assay and results are shown as the fold activation (±s.e.m.) relative to untreated cells from at least three independent experiments. Bottom, 991 and A-769662 increase phosphorylation of endogenous AMPK Thr-172 (pT172) and the AMPK target ACC (pACC) in HEK293 cells (Supplementary Fig. 17). Blots were generated using a capillary-based western blot automated system (Simon, ProteinSimple). (d) HEK293 cells were transfected with myc-α1, FLAG-γ1 and either wild-type GFP-β1, GFP-β1 harbouring a mutation at Ser-108 (S108A) or GFP-β1 lacking the CBM (ΔCBM). Cells were treated with varying concentrations of the 991 activator. Top, complexes were immunoprecipitated using the FLAG-tag antibody and AMPK activity measured using the SAMS peptide assay. Results are shown as the fold activity compared with cells not treated with 991 (±s.e.m. from at least three independent experiments). 991 did not activate AMPK lacking the CBM (ΔCBM, black) in HEK293 cells. Higher concentrations of 991 were required to activate the AMPK β1 Ser-108 to alanine mutant (S108A, grey) in HEK293 cells compared with wild-type (white). Bottom, expression of the GFP-β1 subunit was monitored by western blot analysis in cells treated with and without 10 μM 991. An untransfected sample is included as a control.