eNOS antibody

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	-Rabbit (1:800)

Protocol tips
-Rabbit (1:800)

Publication protocol

Lung tissue (0.1 g) was homogenized in 1 ml of ice-cold lysis buffer containing 1% Nonidet P-40 (NP-40), 0.1% SDS, 0.01 M deoxycholic acid, and a complete protease cocktail inhibitor (Sigma, S8830). The samples were then centrifuged at 13,000 rpm for 20 min at 4°C, and the supernatant was aliquoted and stored at -20°C. Proteins (30 µg) were resolved in 10% SDS-PAGE in reduced condition and electroblotted to a polyvinylidene difluoride membrane (Immobilon-P, IPVH00010; Millipore, Bedford, Mass., USA). After blocking with 5% nonfat milk, the membranes were incubated with polyclonal anti-NOS3 antibody (1:800, GT50505; GeneTex Inc., San Antonio, Tex., USA), anti-NOS2 antibody (1:1,000, PA3-032A; Thermo Fisher Scientific, Vernon Hills, Ill., USA), and anti-NOS1 antibody (1:1,000, PA3-030A; Thermo Fisher Scientific). β-Actin rabbit monoclonal antibody (1:5,000, 13E5; Cell Signaling Technology, Danvers, Mass., USA) was used as an internal control. Goat anti-rabbit IgG (H+L) secondary antibody, HRP linked (1:10,000, 31460; Pierce Biotechnology Inc., Rockford, Ill., USA), was used as a secondary antibody. Densitometric analysis was used to measure the intensity of the target proteins and β-actin using Image J software. The data were normalized to β-actin for each protein.

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