Fig. 4.

991 treatment enhances AMPK activity induced by 5-aminoimidazole-4-carboxamide riboside (AICAR) or C13 in hepatocytes. Hepatocytes were isolated from male C57BL/6NTac mice and cultured overnight as described in materials and methods. Hepatocytes were untreated (control) or treated with AICAR (0.1 mM) or C13 (10 μM) for 1 h in the presence of the indicated concentrations of 991. A: IB analysis was performed with 20 μg lysates using the indicated antibodies. Representative blots are shown; n = 3. Shorter (1 min) and longer (5 min) exposure indicate the exposure time with X-ray film. B--G: AMPK complexes were immunoprecipitated from 50 μg lysates with the indicated antibodies. AMPK activity (in duplicate) was measured in vitro as described in materials and methods. Results are expressed as means ± SE; n = 3. *Significance of AICAR or C13 versus the respective control condition (0, 0.01, or 0.1 μM 991); #significance of 991 versus the respective compound (AICAR or C13) or vehicle. P < 0.05.