Tissue samples of the heart and aorta were homogenized, and the Western blot protocol was performed as previously described [68 (link)]. Membranes were incubated overnight with a primary polyclonal rabbit anti-pan-Akt (1:500, Abcam, ab8805), anti-eNOS (1:1000, Abcam, ab5589), anti-p-eNOS (1:1000, Invitrogen, #PA5-35879), anti-NADPH oxidase 4 (1:2000, Abcam, ab154244), and anti-NF-kappaB p65 (1:1000, Abcam, ab16502) antibodies as well as anti-GAPDH (1:5000, Abcam, ab201822) and anti-β-actin (1:2000, Abcam, ab8227) as a loading control. Antibodies were detected using a secondary peroxidase-conjugated goat anti-rabbit antibody (1:5000, Abcam, ab97051) at room temperature for 2 h. The intensity of bands was visualized using the enhanced chemiluminescence system (ECL, Amersham, UK), quantified using a ChemiDocTM Touch Imagine System (Image LabTM Touch software, BioRad, Hercules, CA, USA), and normalized to GAPDH bands for heart and β-actin bands for the aorta.
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