Fig. 5.

Effects of A-769662, phenformin and AICAR in wild type and double knockout (AMPK-α1^−/−α2^−/−) mouse embryonic fibroblasts (MEFs) and primary mouse hepatocytes. (A) Wild type and double knockout MEFs were analyzed by Western blotting with the indicated AMPK antibodies, or with anti-β-actin as a loading control. In one experiment (second panel from top) AMPK-α1 was immunoprecipitated with anti-α1 antibody prior to Western blotting. (B) Wild type or double knockout MEFs were incubated with the indicated concentrations of A-769662 or with 2 mM phenformin for 1 h and lysates analyzed by Western blotting using the indicated phosphospecific or non-phosphospecific antibodies. Results are presented as means ± SEM of duplicates, and are representative of 2 or more experiments. Blots were quantified by densitometry and phosphorylation of ACC is presented in arbitrary units (AU). (C) Primary hepatocytes from wild type and double knockout mice were incubated with the indicated concentrations of A-769662 or 500 μM AICAR for 4 hr and lysates analyzed by Western blotting using the indicated phosphospecific or non-phosphospecific antibodies.