Anti nrf2 antibody

Heart tissues were homogenized and lysed with RIPA buffer containing 10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease and phosphatase inhibitor cocktails. Lysates were centrifuged at 20,000 g for 40 min at 4 °C and the supernatants were recovered. Total protein concentrations in the supernatants were measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific). For immunoblot analysis, extracted protein samples were separated on 7.5% Mini-PROTEAN TGX precast gradient gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% FBS in Tris-buffered saline plus 0.05% Tween and incubated overnight at 4 °C with an anti-NRF2 antibody (1:1000; Active Motif) and an anti-actin antibody (1:5000; Thermo Fisher Scientific) as a loading control. Primary antibodies were detected with horseradish peroxidase-conjugated species-specific secondary antibodies (GE Healthcare) and ECL plus (Thermo Fisher Scientific) using a LAS 3000 analyzer (GE Healthcare). Immunoblot band intensities were measured using NIH ImageJ software^{63 (link)}. Butylated hydroxyanisole (BHA; Sigma-Aldrich) was administered intraperitoneally to male mice at 8 weeks of age at a dose of 350 mg/kg in corn oil. Uncropped scans of blots are shown in Supplementary Fig. 14a, b, d.

The ChIP assay was performed by using a ChIP-IT High Sensitivity kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocol. A total of 15 million cells were fixed and collected for nuclei isolation. Chromatin was prepared from isolated nuclei, sheared by sonication, and precipitated with anti-NRF2 antibody (Active Motif). Promoter enrichment was quantified by quantitative real-time PCR assay comparing pull-down DNA and input DNA. The sequences of the primer set used are listed as follows: HMOX1.F: 5′-ACA AAG GGA AGG CGG ATT TT-3′; HMOX1.R: 5′-ACT TCC TCC TGC CTA CCA TT-3′; SLC7A11.F: 5′-AGC TTC CCA CAA AGT CGA AG-3′; SLC7A11.R: 5′-ACA TTC CTG CTT GTC TTG GT-3′; GCLC.F: 5′-CGC AGT TGT TGT GAT ACA GCC-3′; GCLC.R: 5′-GGA CTG AGA CTT TGC CCT AAG AA-3′; GCLM.F: 5′-ATT CCA AAC TGA GGG AGC TGT TT-3′; 5′-GCLM.R: ATG AGT AAC GGT TAC GAA GCA CT-3′; NQO1.F: 5′-GTG TGA CAG AGG CCT CAA AA-3′; NQO1.R: 5′-TGA TCC CTG GAC TCT CTT GG-3′.