Fig. 5.

991 treatment enhances AMPK activity induced by AICAR in C₂C₁₂ muscle cells. A: IB analysis was performed with 20 μg lysates from C₂C₁₂ myoblasts or myotubes with the indicated antibodies. Representative blots are shown; n = 2 for each condition. Creatine kinase was used as a differentiation marker. B: IB analysis was performed with 20 μg lysates from C₂C₁₂ myotubes untreated (control) or treated with AICAR (2 mM) in the presence or absence of the indicated dose of 991 for 30 min. Representative blots are shown; n = 3 for each condition. C: schematic representation of the sequential immunoprecipitation protocol for the isolation of AMPKγ3 and AMPKγ1 from tissue lysates (top). AMPKγ3 and AMPKγ1 immune complexes were sequentially immunoprecipitated from 200 μg mouse gastrocnemius muscle lysate protein. IB analysis was then performed on the input (10% = 20 μg), the SN (10% = 20 μg), and the immunoprecipitates (IP) from the first and second immunoprecipitation using the indicated antibodies (bottom). D: AMPKγ3 and AMPKγ1 activity (in duplicate) from 200 μg C₂C₁₂ myotube lysate protein were measured after sequential immunoprecipitations as described in materials and methods. Results are expressed as means ± SE; n = 3. *Significance of AICAR versus the respective control condition (0 or 10 μM 991); #significance of 991 versus the respective control (0 or 2 mM AICAR). P < 0.05.