Horseradish peroxidase conjugated species specific secondary antibodies

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.

Whole-cell lysates of MSC were collected, separated by SDS–PAGE and transferred onto polyvinylidene difluoride membrane by liquid transfer method. Blots were blocked in 10% skim milk (Bio-Rad, Hercules, CA, USA) in tween 0.1% tris-buffered saline (TTBS) 1 × 1 h at room temperature. The primary antibodies used were a mouse anti-lamins A/C 1:200 (Millipore, JOL2), a rabbit anti-prelamin A 1/100 (ANTOO45, Diatheva) and a β-actin 1/200000 (Sigma-Aldrich). Membranes were incubated during the night at 4 °C. Antigen–antibody binding was detected using horseradish peroxidase-conjugated species-specific secondary antibodies (GE-Healthcare, Little Chalfont, UK) followed by enhanced chemiluminescence western blotting detection reagents (Perkin-Elmer, Waltham, MA, USA).