Western blot analysis.

Equal amounts of total protein were loaded in each well of precast gels (Bio-Rad Laboratories). All samples from each subject were loaded on the same gel. Proteins were separated according to their molecular weight by SDS-PAGE and semi-dry transferred to a PVDF membrane (Bio-Rad). The membranes were blocked in either 2% skim milk or 3% BSA in TBS, including 0.1% Tween-20 (TBST) before an overnight incubation in primary antibody at 4°C and a subsequent 1-h incubation in horseradish peroxidase-conjugated secondary antibody at room temperature. The bands were visualized with ECL (Millipore) and recorded with a digital camera (ChemiDoc MP Imaging System, Bio-Rad Laboratories). Densitometry quantification of the Western blot band intensity was done using Image Lab version 4.0 (Bio-Rad Laboratories) and determined as the total band intensity adjusted for background intensity. Representative blots are shown in Fig. 2.

Representative Western blots, including the molecular weight of band migration. 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; ACCβ Ser-221 phos, acetyl-CoA carboxylase β serine-221 phosphorylation; AMPKα2, AMP-activated protein kinase α2; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; eEF2, eukaryotic elongation factor 2; FXYD1, phospholemman; mTOR, mammalian target of rapamycin; PKCα/β Thr-638/641 phos, protein kinase Cα/β threonine 638/641 phosphorylation; p70S6K1, ribosomal protein S6 p70 kinase 1; PLN, phospholamban.