Fig. 7. EtOH and PRAS40 knockdown increase AMPK activity toward raptor.

C2C12 myocytes or PRAS40 KD cells were pre-incubated for 1 h in the presence or absence of the AMPK inhibitor compound C (20 μM). Knockdown cells were harvested, while scrambled control cell were further treated with EtOH. The specificity of compound C was examined via Western blot using anti-phospho-raptor. Results were normalized to total protein and expressed as a percentage of scrambled control levels (panel A). Panel B, an in vitro AMPK activity assay was performed where raptor was utilized as the substrate in the presence of AMP. Cells were lysed in 1% NP-40 and AMPK was immunoprecipitated from 150 μg of lysate. The activity was determined in the presence or absence of 20 μM compound C as described above. Results were normalized with immunoprecipitated AMPK which was assessed by immunoblotting. Each bar graph represents mean ± SE of 3 independent experiments consisting of 4 replicate samples per experiment. Groups with different letters are significantly different from one another (* P< 0.05). Groups with the same letters are not significantly different.