Protein samples from the thoracic aorta were prepared in lysis buffer as described previously [11 (link), 41 (link)–47 (link)]. The proteins (15 μg) were separated by SDS–PAGE and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was then incubated overnight (4 °C) with a primary antibody against Collagen I (1:1000, Sigma–Aldrich), elastin (1:200, Santa Cruz Biotechnology), TGFβ−1(1:100, Santa Cruz Biotechnology), scleraxis (1:100, Santa Cruz Biotechnology), MMP-2, MMP-9 (1:1000, EMD Millipore Corporation), Klotho (R&D Systems), and β-actin (1:10,000, Abcam). Goat anti-mouse or goat anti-rabbit conjugated with horseradish peroxidase (1:3000–1:5000, Santa Cruz Biotechnology) was used as a secondary antibody and incubated for 1 h at room temperature. Specific proteins were detected by chemiluminescent methods using Amersham™ ECL™ Western blotting detection reagents (GE Healthcare, UK). Protein abundance on Western blots was captured using Chem-Doc (Bio-Rad) and quantified by densitometry using the Image lab software (Bio-Rad, Hercules, CA).