Figure 5. Increased activation by 991 is mediated by the N-terminal region of γ2.

(A) A schematic representation of the γ1- and γ2-isoforms is shown, together with the two chimeric γ-constructs [γ(C1) and γ(C2)] used in the present study. Different regions within the isoforms are shown colour-coded. The conserved core region, containing the four CBS repeats, is indicated in red for γ1 and purple for γ2. The N-terminal region of γ1 is shown in green, the N-terminal sequence for γ2b (the short isoform of γ2) is shown in yellow and the long N-terminal extension in γ2a (the long isoform of γ2) is coloured in blue. All constructs have a C-terminal hexa-His tag shown in grey. (B) Expression of the two chimeric proteins was confirmed by western blotting using an anti-His antibody and is shown relative to γ1-expression. Vinculin expression was used as a loading control. (C) β1β1 dKO cells were transfected with different AMPK complexes and treated with DMSO or 991 (1 μM) for 30 min. Thr¹⁷² phosphorylation and total α expression levels were determined by fluorescence-based western blotting. Although the α2β2γ2-complex was analysed in the same experiment at the same time, it was performed on a separate blot and is therefore shown as a distinct panel. (D) Blots were quantified and the increase in Thr¹⁷² phosphorylation in response to 991 relative to the DMSO control was determined as a fold change. Results are for three independent experiments, performed in duplicate, and are plotted as means ± SEM. Statistically significant results, determined by one-way ANOVA, using Bonferroni correction, are indicated (*P < 0.05).