Figure 2.

AMPK activity markers and ATP/ADP ratios in C2C12 myotubes in response to AMP-dependent and AMP-independent activators. A–C, C2C12 myotubes were treated with mitochondrial inhibitors: rotenone (2 μg/ml), phenformin (5 mm), or oligomycin (100 ng/ml) for 30 min. A, Western blotting of AMPKα-phospho-Thr-172, ACC-phospho-Ser-79, and AMPKβ (loading control). B, AMPK-specific activity was assessed by the SAMS kinase assay, measured as picomole of [γ-³²P]ATP/μg of cell protein/min and expressed as % untreated control. C, ATP/ADP ratios in C2C12 myotubes were measured by a luciferase/luciferin bioluminescence assay and expressed as % untreated control. D and E, C2C12 myotubes were treated with 991 (1 or 10 μm) for 30 min. D, Western blotting was performed as previously described. E, AMPK-specific activity was measured as previously described. F, ATP/ADP ratios were measured as previously described. All Western blots are representative of 3 or 4 biological replicates. Bar graphs indicate mean ± S.D. of 3 biological replicates. Statistical analysis was performed by one-way ANOVA with Dunnett's multiple comparison post-test comparing all treatments to untreated controls; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.