Ab201822

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.

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).

Total protein lysates were prepared from rat heart tissues using protein extraction reagent (USA, Thermo, 78510) as described previously.²¹, ²³ A mitochondria/cytosol fractionation kit (UK, Abcam, ab65320) was used for isolation of mitochondrial and cytosolic fractions from cardiac tissues, following the instructions in the manual.²⁴ Protein extracts were first separated through SDS‐PAGE and then transferred to nitrocellulose membranes. The membranes were incubated at 4℃ overnight with appropriate primary antibodies (ISCA1 [USA, Thermo, PA5‐60121]; TOMM20 [USA, Thermo, PA5‐52843]; NDUFA9 [UK, Abcam, ab14713]; NDUFS3 [UK, Abcam, ab110246]; SDHB [UK, Abcam, ab14714]; GAPDH [UK, Abcam, ab201822]) and subsequently kept for 1 h at RT with the appropriate secondary antibodies. Western blot images were acquired (Bio‐Rad, ChemiDoc XRS+Gel Imaging System, USA) and quantitatively analyzed with Image J software.