NRCMs or cardiac tissues were lysed in lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1% SDS, and 0.5% sodium deoxycholate] plus phenylmethylsulfonyl fluoride (PMSF) (Solarbio) on ice for 20 min. Then, the samples were centrifuged at 12,000 rpm at 4°C for 15 min to obtain the protein extract. After the protein concentration was quantitated by bicinchoninic acid (Thermo Fisher Scientific), 2 μg of appropriate primary antibody and protein A–Sepharose (Amersham Biosciences) were added to the protein samples with gentle shaking at 4°C for 24 hours, followed by centrifugation at 5000 rpm at 4°C for 10 min. The pellet was washed twice with wash buffer I [50 mM tris-HCl (pH 7.5), 500 mM sodium chloride, 0.1% NP-40, and 0.05% sodium deoxycholate] and once with wash buffer II [50 mM tris-HCl (pH 7.5), 0.1% NP-40, and 0.05% sodium deoxycholate]. Bound proteins were eluted by boiling beads in 2× sample buffer, and the precipitated proteins were subjected to SDS-PAGE. Immunoblot data were quantified and analyzed with a Gel-Pro 4.5 analyzer (Media Cybernetics, USA).

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