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Fig. 6. BMP4 stimulation induces Smad4/Smad5/CBP association and DNA-binding activity in spermatogonia. (A) Eighty µg of total cell protein extracts from control or BMP4-treated spermatogonia were immunoprecipitated for Smad4 (lower panel) and immunoblotted for Smad5 and CBP (upper and middle panel, respectively). (B) DNA-binding activity in nuclear extracts from control and BMP4-treated spermatogonia probed with a 3xGCCG-[{alpha}-32P]ATP double-stranded oligonucleotide, in the presence of 100x molar excess of competitors (wild-type or mutated 3xGCCG double-stranded oligonucleotides) or in the presence of an anti-Smad4 antibody. (C) DNA-binding activity in nuclear extracts from control and BMP4-treated spermatogonia probed with a 3xGCCG-[{alpha}-32P]ATP double-stranded oligonucleotide, in the presence or absence of anti-Smad5 and anti-Smad8 antibodies.





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