Fuji las3000 densitometer

Hearts or coronary arterioles were homogenized in lysis buffer (Cellytic MT Mammalian Tissue/ Lysis/extraction Reagent; Sigma). Protein concentrations were measured using a BCA Protein Assay Kit (Pierce) and equal amounts of protein (10, 20, or 40 μg) separated by SDS-PAGE and transferred onto nitrocellulose membranes. Protein expression was detected using the appropriate primary antibody: TNF-α (1:500; R&D Systems), IL-6 (1:500; Abcam, Inc.), SOD2 (1:1,000; EMD Chemical), eNOS (1:100; Santa Cruz Biotechnology), p-eNOS (1:100; Santa Cruz Biotechnology), β-actin (1:2,000; Abcam Inc.), and corresponding secondary antibodies (1:1,000~2,000 dilution). Signals were enhanced by chemiluminescence (ECL; Amersham) and visualized with a Fuji LAS3000 densitometer. The density of protein bands obtained from the images was analyzed using Multigauge software (Fuji film). The relative densities were calculated and normalized to those of the corresponding internal reference β-actin, and then normalized to the corresponding WT control mice, which was set to a value of 1.0.