Las 3000 analyzer

The cells were lysed in buffer containing 10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, complete protease inhibitor cocktail, and PhoStop EASYpack (Roche). After centrifugation at 15,000 rpm for 20 min at 4 °C, the supernatants were recovered and total protein concentrations were measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific). For immunoblot analysis, extracted proteins were separated on 4–15% Mini-PROTEAN TGX precast gradient gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with 2% nonfat dry milk powder in Tris-buffered saline plus 0.05% Tween and incubated overnight at 4 °C with an anti-MYLK3 antibody (1:500; Novus Biologicals, Littleton, CO, USA; cat# NBP1–86648) recognizing residues 27−144 of human cMLCK and an anti-β-actin antibody (1:5000; Sigma cat# A5441) as a loading control. Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies (GE Healthcare) and ECL plus (Thermo Scientific) using a LAS 3000 analyzer (GE Healthcare). Human cardiac tissue and non-transfected HEK293T cells were used as controls. Immunoblot band intensities were measured using ImageJ software (National Institutes of Health).

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.