Fig. 7. Nuclear extracts from MDCK-Snail cells contain specific Ets-1 and Sp-1 binding complexes not observed in MDCK-CMV cells. (A) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed by EMSA showing the binding of nuclear proteins to the HA-SP-1 and Ets-1 elements. Nuclear proteins from serum free cultures were incubated with the ³²P-labelled wild-type probe encompassing the sequence from nts -85 to -75 of MMP-9 promoter in the absence or presence of 500-fold molar excess of wild-type (lanes 3 and 8) or mutant cold oligonucleotides (lanes 6 and 10). Lanes 1 and 2 show EMSA in which the nuclear extracts were not added. The ³²P-labelled mutant probe was used in lanes 5 and 9. Black arrowhead indicates the main complex detected. The complete sequence of the wild-type probe is shown at the bottom of the figure in which the mutated nucleotides are underlined. The gel shown is representative of at least two independent experiments. (B) Nuclear extracts of MDCK-CMV (lane 2) and MDCK-Snail cells (lanes 3-6) were analysed in band-shift assays. The ³²P-labelled wild-type (wt) oligonucleotide encompassing the sequence from nts -97 to -75 of the MMP-9 promoter is shown at the bottom. Nuclear extracts were incubated with either an anti-Sp-1 (lane 4) or anti-Ets-1 (lane 5) antibodies, or control rabbit IgG (lane 6). A black arrowhead indicates the main complex detected, the intensity of which is lowered by both antibodies. The gel shown is representative of four independent experiments.