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. 2011 Aug;85(15):7836–7848. doi: 10.1128/JVI.01197-10

Fig. 9.

Fig. 9.

EMSAs showing Smad4 competing with ZEB1 for binding to Zp or complexing with ZEB1 on the ZV element. (A) Protein competition EMSA with E. coli-expressed Smad4 and ZEB1. EMSAs were performed with 8 ng of radiolabeled double-stranded SBE2-WT probe corresponding to the nt −26 to −6 region of Zp. 6×His-tagged-Smad4 purified from E. coli and an extract from 293T cells that had been transfected with a ZEB1 expression plasmid were used as the protein sources. Lane 1, no protein; lanes 2 and 4, 225 ng of 6×His-Smad4; lane 3, 225 ng of 6×His-Smad4 plus 3 μg of antibody specific to Smad4; lane 5, 2.9 μg of ZEB1-containing extract plus 3 μg of antibody specific to ZEB1; lanes 6 to 9, 2.9 μg of ZEB1-containing extract plus 0, 25, 75, and 225 ng of 6×His-Smad4, respectively. (B) Protein competition EMSA with activated Smad4 and ZEB1. EMSAs were performed with radiolabeled SBE2-WT DNA as the probe, activated, nontagged Smad4 purified from 293T cells cotransfected with expression plasmids for Alk5 CA, and Smad4 and the ZEB1-containing extract used for panel A as the protein sources. Lane 1, no protein; lane 2, 200 ng of activated Smad4 plus 3 μg of antibody specific to Smad4; lane 3, 200 ng of activated Smad4; lane 4, 2.9 μg of ZEB1-containing extract plus 3 μg of antibody specific to ZEB1; lanes 5 to 9, 2.9 μg of ZEB1-containing extract plus 0, 100, 200, 500, and 1,000 ng of activated Smad4, respectively. (C) EMSAs showing the formation of Smad4/ZEB1 complex bound to the ZV element of Zp. EMSAs were performed with 8 ng of radiolabeled SBE2-WT DNA or variants of it containing 2-bp substitution mutations in either the SBE2 or the ZV element. Protein sources were the same ones used for panel B. Lanes 1, 7, and 13, no protein; lanes 2, 10, and 14, 0.5 μg of activated Smad4 plus 3 μg of antibody specific to Smad4; lanes 3, 11, and 15, 0.5 μg of activated Smad4; lanes 4, 8, and 16, 2.9 μg of ZEB1-containing extract plus 3 μg of antibody specific to ZEB1; lanes 5, 9, and 17, 2.9 μg of ZEB1-containing extract; lanes 6, 12, and 18, 0.5 μg of activated Smad4 plus 2.9 μg of ZEB1-containing extract. Locations of the protein/DNA complexes are indicated.