NF‐κB and Nrf2 activation assay

Cardiac and renal NF‐κB and Nrf2 activation was assessed by measuring the p65 (canonical NF‐κB subunit) and Nrf2 contained in tissue nuclear versus cytoplasmic fraction using TransAM^® NF‐κB (p65) and TransAM^® Nrf2 assay kits (Active Motif). The aliquots of the cryopulverized myocardium or kidney cortex were homogenized (Potter‐Elvehjem Tissue Grinder with Teflon Pestle, 2 cm³ size, Wheaton Science Products, Millville, NJ) in ice‐cold 1× Hypotonic Buffer containing protease and phosphatase inhibitors, detergent and dithiothreitol. Tissue nuclear and cytoplasmic fractions, and the nuclear extracts were prepared in line with the manufacturer's instructions. The wells of a respective 96‐well microplate were loaded with 10 μL of the tissue nuclear extract or the tissue cytoplasmic fraction, each containing 20 μg of protein in the NF‐κB (p65) assay and 10 μg of protein in the Nrf2 assay. The absorbance of the wells was read (Microplate Reader, Thermo Lab Multiscan RC, model No 351) versus blank wells containing 10 μL of the Complete Lysis Buffer. Data shown are the results from wells assayed in duplicate.