4.11. Western Blotting of PI3K, Akt, mTOR, TGF-β1, and Caspase-3

Proteins were extracted from frozen heart tissue and the protein concentration was determined by a BCA (bicinchoninic acid) Protein Assay Kit (P0010, Beyotime, Beijing, China). Equal amounts of loaded protein were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to polyvinylidene difluoride membranes, which were then blocked at room temperature for 1 h with 5% dry skim milk in Tris-buffered saline containing 1% Tween-20. The membranes were reacted with antibodies for the glucocorticoid receptor, PI3K (1:1000), Akt (1:1000), p-Akt (1:1000), mTOR (1:1000), p-mTOR (1:1000), TGF-β1 (1:1000), and GAPDH (1:1000) overnight at 4 °C. The membranes were washed thoroughly. PI3K, Akt, p-Akt, mTOR, p-mTOR, TGF-β1, and caspase-3 incubated with antirabbit IgG secondary antibody were conjugated to horseradish peroxidase for 1 h (1:5000), and then washed thoroughly. The protein bands were detected by chemiluminescence reagents (ECL kit, Amersham Biosciences, Beijing, China). A digital imaging system (ImageQuant LAS 500, GE Healthcare Life Sciences, Beijing, China) was used to visualize protein bands, and densitometry was performed with ImageJ software. The density of each immunoreactive band was normalized to the density of the corresponding GAPDH band.