2.6. Western blot analysis

Snap‐frozen hearts were homogenized and lysed using ceramic beads in Precellys 24 (5000 rpm, 2 × 10 s‐5 s) to extract nuclear and cytosolic proteins (Nuclear Extract protocol, Active Motif). Protein concentration was calculated using a Bradford assay (Bradford reagent, Sigma‐Aldrich). Cytosolic proteins (30 µg) were separated by 8–10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Next, membranes were blocked for 1 h at room temperature with 5% nonfat milk in TRIS‐buffered saline (TBS) with Tween 20 (0.1%). Membranes were then incubated overnight at 4°C with the following primary antibodies in TBS‐Tween 20–5% nonfat milk: ^Ser473phospho‐Akt, ^Tyr1150phospho‐IRβ, Akt1 (1:1000, Millipore), Irβ (1:1000, Santa Cruz Biotechnology), and α‐tubulin (1:2000, Santa Cruz Biotechnology). Membranes were then incubated for 1 h at room temperature with the appropriate horseradish peroxidase‐conjugated anti‐IgG (1:5000, Jackson ImmunoResearch). Proteins were visualized by enhanced chemiluminescence with the Western Blot ECL substrate (Clarity, Bio‐Rad) and video acquisition (chemidoc‐xrs‐system, Bio‐ Rad). Relative amount of protein was quantified by densitometry (Image Lab, Bio‐Rad) and expressed as a ratio of the appropriate loading control. Phosphorylated proteins were expressed relative to total protein, and nonphosphorylated proteins were expressed relative to tubulin. Finally, results were expressed as the phosphorylated‐to‐total ratio relative to LFD‐NaCl or HFD‐NaCl groups.