Samples of the left ventricle were homogenized. Western blot analysis was performed as described elsewhere [44 (link)]. The membranes were incubated overnight with primary polyclonal rabbit anti-NADPH oxidase 4 (1:2000, Abcam, ab154244) and anti-GAPDH (1:5000, Abcam, ab201822) as loading controls. The 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 the bands was visualized using an enhanced chemiluminescence system (ECL, Amersham, UK), quantified using a ChemiDocTM Touch Imagine System (Image LabTM Touch software, version 5.2 build 14, 11 September 2014, BioRad, Hercules, CA, USA), and normalized to GAPDH bands.
The concentration of CD was measured in the lipid extracts of the left ventricle homogenates as previously described [45 (link)]. Briefly, after chloroform evaporation under an inert atmosphere and the addition of cyclohexane, the CD concentration was determined spectrophotometrically (λ = 233 nm, GBC 911A, Bio-Rad Laboratories GesmbH, Wien, Austria).
The concentration of CD was measured in the lipid extracts of the left ventricle homogenates as previously described [45 (link)]. Briefly, after chloroform evaporation under an inert atmosphere and the addition of cyclohexane, the CD concentration was determined spectrophotometrically (λ = 233 nm, GBC 911A, Bio-Rad Laboratories GesmbH, Wien, Austria).
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