Whole-cell protein was extracted using radioimmunoprecipitation assay lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Proteins were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% nonfat milk and then incubated with primary antibodies and secondary antibodies.

Nuclear fractions for IP were prepared by ammonium sulfate precipitation. Briefly, cells were resuspended in buffer A [10 mM Hepes (pH 7.6), 10 mM KCl, 25 mM 10% glycerol, 1 mM dithiothreitol (DTT), and 1 mM PMSF] for 5 min on ice. Nuclei were harvested by centrifugation (1300g for 4 min) and lysated by 0.3 M ammonium sulfate in buffer C [10 mM Hepes (pH 7.6), 3 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 1 mM PMSF]. Soluble nuclear proteins were separated by ultracentrifugation (100,000g for 30 min) and precipitated with ammonium sulfate (0.3 g/ml) for 30 min on ice. Protein precipitate was isolated by ultracentrifugation (100,000g for 30 min) and resuspended in IP lysis buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 1 mM DTT, and 1 mM PMSF] for IP or gradient sedimentation.

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