Anti elastin

Arterial tissue (carotid arteries and thoracic aorta) was collected and snap-frozen in liquid nitrogen before homogenization in modified RIPA lysis buffer containing 1% SDS and 1% Triton. For collagen extraction, tissue was first subjected to acid hydrolysis with 6N HCl at 100°C for 72 hours, then homogenized in RIPA lysis buffer containing 1% SDS and 1% Triton. Equal amounts of protein were separated by SDS-PAGE and transferred to Trans-Blot® Turbo^™ Mini-size PVDF membranes (Biorad, Hercules, CA). Primary anti-CYP4A1 (1:200, Santa Cruz, Dallas, TX), anti-MMP12 (1:250, Santa Cruz, Dallas, TX), anti-MMP2 and -MMP9 (1:1000, Millipore, Temecula, CA), anti-MMP3, -MMP7 and – MT1-MMP (1:1000, Abcam, Cambridge, MA), anti-TIMP1, -TIMP2, -TIMP3 and – TIMP4 (1:1000, Abcam), anti-collagen I and – collagen III (1:1,000, Abcam), and anti-elastin (1:200, Santa Cruz, sc-58756, recognizes intact elastin and degradation products) and secondary anti-rabbit or anti-mouse antibodies (BioRad), as appropriate, were used for Western blotting. Bands were visualized by the Odyssey Western blot detection system (Azure Biosystems, Dublin, CA). Bands were quantified using Un-Scan-It Image software (Silk Scientific Corporation, Orem, UT). Data are normalized to β-tubulin (loading control).