Homogenization of heart tissues was carried out in a 10-fold volume of ice-cooled phosphate-buffered saline (PBS) (ice-cooled, pH 7.4). Following centrifugation at 10,000× g for 20 min and 4 °C, the supernatant was then collected for analysis of oxidative and inflammatory markers. ELISA kits were used in the assessment of the hearts’ content of malondialdehyde (MDA), reduced glutathione (GSH), enzymatic activities of superoxide dismutase (SOD), catalase (CAT) (Cat. No. MD 2529, GR 2511, SD 2521, and CA 2517, Biodiagnostic, Giza, Egypt, respectively), and protein carbonyl, interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) (Cat. No. ab238536, ab234570, and ab100785, Abcam, Cambridge, UK, respectively).
Nuclear fractions of tissue homogenates were obtained using NE-PER nuclear and cytoplasmic extraction kit (Cat. No. 78833, Thermo Fisher Scientific, Waltham, MA, USA). Protein content of the nuclear extracts was determined, and a volume equivalent to 80 μg was employed in the assessment of the DNA-binding activity of NF-kB p65 using NF-κB p65 ELISA Kit (Cat. No. ab133112, Abcam, Cambridge, UK). Results are expressed as fold change of control.
Nuclear fractions of tissue homogenates were obtained using NE-PER nuclear and cytoplasmic extraction kit (Cat. No. 78833, Thermo Fisher Scientific, Waltham, MA, USA). Protein content of the nuclear extracts was determined, and a volume equivalent to 80 μg was employed in the assessment of the DNA-binding activity of NF-kB p65 using NF-κB p65 ELISA Kit (Cat. No. ab133112, Abcam, Cambridge, UK). Results are expressed as fold change of control.