Goat monoclonal anti cd31

Lysates (30-50 μg) were prepared from the carotid artery, aorta or VSMCs, subjected to SDS-polyacrylamide gel (10%) electrophoresis and transferred to a PVDF membrane (Perkin Elmer Life Sciences, Boston, MA) [16 ]. Antibodies used include a mouse monoclonal anti-nestin (1:500; Chemicon), mouse monoclonal anti-eNOS (1:500; BD Bioscience), goat monoclonal anti-CD31 (1:500; SantaCruz Biotechnologies), rabbit polyclonal anti-caldesmon (1:2500; Abcam), rabbit polyclonal anti-smooth muscle-22α (1:5000; Abcam), rabbit polyclonal anti-smooth muscle α-actin (1:5000; Abcam), and mouse monoclonal anti-GAPDH (1:50,000; Ambion, Austin TX). Following overnight incubation at 4°C, the appropriate secondary antibody-conjugated to horseradish peroxidase (1:20,000, Jackson Immunoresearch, West Grove, PA) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software® and the target protein signal was depicted as arbitrary light units normalized to GAPDH protein levels.

Lysates (30–50 μg) prepared from the left ventricle of normal and suprarenal aorta constricted rats, adult and neonatal ventricular fibroblasts and rat microvascular endothelial cells were subjected to SDS-polyacrylamide gel (15%) electrophoresis and transferred to a PVDF membrane (Perkin Elmer Life Sciences, Boston, MA).[6 (link),14 (link)] Antibodies used include mouse monoclonal anti-nestin (~240 KDa;1:500; EMD Millipore, Darmstadt, Germany), goat monoclonal anti-CD31 (~140 kDa; 1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), mouse monoclonal anti-smooth muscle α-actin (~43 kDa; 1:5000; Sigma-Aldrich), rabbit polyclonal anti-collagen type I (~240 KDa pro-collagen;1:1000; Abcam, Cambridge, MA), rabbit polyclonal anti-prolyl 4-hydroxylase alpha polypeptide III (~75 KDa;1:500; EnoGene, New York, NY) and a mouse monoclonal anti-GAPDH (~37 kDa; 1:50,000; Ambion, Austin TX). Following overnight incubation at 4°C, the appropriate secondary antibody-conjugated to horseradish peroxidase (1:20,000, Jackson Immunoresearch, West Grove, PA) was added and bands visualized utilizing the ECL detection kit (Perkin Elmer). Films were scanned with Image J software^® and the target protein signal was depicted as arbitrary light units normalized to GAPDH protein.