Protein expression in thoracic aortas was determined by immunoblotting, as described previously [23 (link)]. Briefly, thoracic aortas were homogenized with T-PER tissue protein extraction reagent (Thermo-Pierce, 78510, Rockford, IL, USA) containing protease inhibitor cocktails (Sigma-Aldrich, P8340) and protein concentrations were determined by BCA Protein Assay Kit (Bio-Rad, 5000001, Hercules, CA, USA). Proteins from each sample (20 μg) were separated by 8%–12% Tris SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., 1620256, CA, USA). After blocking with 5% non-fat dry milk, membranes were incubated with anti-p-Akt (Ser473), anti-p-S6 Ribosomal Protein (Ser235/236), anti-Akt (C73H10), anti-S6 Ribosomal Protein (Cell Signaling Technology, 4058; 4856; 2938; 2217, Beverly, MA, USA), or anti-β-actin (AC-15) (Sigma-Aldrich, A5441) antibodies overnight at 4 °C, and incubated with the secondary antibodies (Santa Cruz, sc-2004; sc-2005, Santa Cruz, CA, USA) at room temperature for 1 h. Proteins were visualized with enhanced chemiluminescence reagents (Thermo-Pierce, Rockford, IL, USA) and the blots were exposed to hyper film. Results were quantified by using Quantity-one software (Bio-Rad).