NRCMs and HEK293T cells were lysed in lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate] with PMSF (phenylmethylsulfonyl fluoride) (Solarbio) and protease inhibitor cocktail (Roche, Basel, Switzerland) on ice for 20 min. Lysate was cleared by centrifugation at 12,000 rpm at 4°C for 15 min to obtain the cell extracts. The protein concentration of the cell extracts was quantitated by bicinchoninic acid (Thermo Fisher Scientific). Immunoprecipitation was performed as described previously (40). Briefly, cell extracts were incubated with the appropriate primary antibody (2 μg) and Protein A Sepharose (Amersham Biosciences/GE Healthcare, Chicago, IL, USA) at 4°C for 12 hours, followed by centrifugation at 3000 rpm at 4°C for 10 min. Pellets were 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 using 8 or 10% gels followed by Western blot analysis. Immunoblot data were quantified and analyzed with a Gel-Pro 4.5 Analyzer (Media Cybernetics).

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