Figure 5.

AMP-activated protein kinase (AMPK) regulates protein anabolism during exposure to high CO₂ in cultured myotubes. (A) AMPK phosphorylation (pAMPK) in skeletal muscle from mice exposed to high CO₂ (HC) versus room air (NC) for 60 days. Actin was used as a lane loading control (n = 3). (B) pAMPK in primary myotubes exposed to 40 mm Hg (NC) or 120 mm Hg (HC) CO₂ for 24 hours. Actin was used as a lane loading control (n = 3). (C) pAMPK in C2C12 myotubes exposed to 40 mm Hg (NC) or 120 mm Hg (HC) CO₂ for different durations. Actin was used as a lane loading control. (D) Myotubes were transfected with scrambled (Scr), AMPKα1, or AMPKα2 siRNA and then exposed to normal (NC) or high CO₂ (HC). Representative figures depict results of qPCR with primers to amplify 45S pre-RNA/GAPDH. Cell samples were processed for Western blotting and probed with specific antibodies to indicate AMPKα1 or AMPKα2 siRNA expression as a control of transfection and silencing efficiency in each condition (n = 3). (E) Conditions similar to D with myotubes exposed to NC and HC for 24 hours in the presence of puromycin-containing medium. Samples were then lysed and immunoblotted with antipuromycin antibody, and the intensity of the smears was scored as shown. Membrane was then stripped and probed with actin as a lane loading control. Transfection control was carried out by Western blotting and probing with specific antibodies to indicate specific silencing of AMPKα1 or AMPKα2 siRNA (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001.