Electrophoretic mobility shift assays were performed as follows. Mixtures of purified Smad1 (10 ng) protein and 0.5 ng of carboxyfluorescein (FAM)–labeled probes were incubated at room temperature for 30 min in a 10-μl reaction volume. The reaction buffer contained 25% glycerol, 50 mM KCl, 0.5 mM EDTA, 10 mM DTT, and 5 mM tris-HCl and had a pH of 8.0. The mixtures were resolved on a 6% nondenaturing polyacrylamide gel (60:1 acrylamide-to-bisacrylamide ratio) containing 5% glycerol in 0.5× tris-borate EDTA buffer. The gels were visualized using a Typhoon FLA9500 Scanner.

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