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For binding assays, excess His-tagged p65 protein was incubated with FLAG-tagged p53 proteins in binding buffer (150 mM NaCl, 20 mM Hepes at pH 7.9, 0.1% NP40) for 2 h at 4 °C. The protein complexes were then incubated with M2 agarose for 30 min at 4 °C. Beads were washed five times with wash buffer (20 mM Tris-HCl at pH 7.9, 20% glycerol, 0.1 mM EDTA, 150 mM KCl, 0.1% NP40). Bound proteins were eluted in sample buffer and analyzed by immunoblot.
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