Western Blotting for Measuring MMP-2, MMP-9, Fibronectin, TIMP-2, Caspase-3, and eNOS

Western blotting was conducted as previously described.^16,41 Briefly, the genomic DNA in the whole cell lysates was sheered via sonication on ice three times (10 s each). The whole cell lysates were loaded onto SDS polyacrylamide gels for electrophoresis separation. The separated protein bands were blotted onto Immun-Blot PVDF membranes (Bio-Rad) for detection with the following primary antibodies (Abs): mouse anti-MMP-2 (cat#MAB3308, Millipore), rabbit anti-MMP-9 (cat#AB13458, Millipore), rabbit anti-fibronectin (cat#AB1954, Millipore), rabbit anti-TIMP-2 (cat#AB2965, Millipore), rabbit anti- caspase-3 (cat# 9665, Cell Signaling), rabbit anti-eNOS (cat# 610298, BD Bioscience) and mouse anti-GAPDH (cat#CB1001, Calbiochem) as a loading control. The primary antibody bindings were detected with the corresponding secondary Abs conjugated with horse-radish peroxidase (HRP) (Amersham GE Healthcare) and visualized using the standard enhanced chemiluminescence (ECL) detection (Amersham GE Healthcare). Western blotting results were quantified by densitometric measurement using ImageJ (National Institutes of Health, Bethesda, MD).