Fig. 1. Adenosine activates AMPK in IEC-6 cells. (A) IEC-6 cells were incubated either in the presence or absence of 10 µM adenosine (Ado) or 500 µM AICAR (Aicar) for 10 minutes and 1 hour, respectively; C, control. AMPK complexes were isolated from cell lysates by immunoprecipitation with antibodies to either anti-1AMPK (open bars) or anti-2AMPK antibody (solid bars). 1AMPK and 2AMPK complex activities were measured by phosphorylation of AMARA peptide. Activities shown are the mean and s.e. of five and three independent experiments for 1AMPK and 2AMPK, respectively, with each assayed in duplicate. The statistical significance of the adenosine and AICAR effects was established by Student's t-test (**P<0.01; ***P<0.001). (B) Cells were treated with either adenosine or AICAR or control (Ctrl), as detailed above. Equal amounts of protein (40 µg) were loaded to allow direct comparisons of phosphorylated AMPK (P-AMPK) levels. ß-actin was used as a loading control. A representative western blot is shown.