Fig. 4. Nuclear extracts from MDCK-Snail cells did not bind to the putative E-box element of the MMP-9 promoter but bound to the E-pal element of E-cadherin promoter. (A) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed in band-shift assays using the ³²P-labelled E-box wild-type probe of the MMP-9 promoter containing the putative E-box at nt -648 (lanes 2-8), or the E-pal element of the mouse E-cadherin promoter (lanes 1, 9-11). Lanes 1 and 2 show EMSA in which the nuclear extract was not added. The retarded complexes were detected when using the E-pal probe of the E-cadherin promoter and are indicated by a black arrowhead. Incubation of the MDCK-Snail nuclear extracts in the presence of control rabbit IgG or an anti-Snail antibody is shown in lanes 10 and 11, respectively. The complete sequence of the E-box MMP-9 probe and the E-pal E-cadherin probe are shown at the bottom of the figure with position of E-boxes underlined. The gel shown is representative of at least two independent experiments. (B) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed in band-shift assays using as a probe the E-pal element of the E-cadherin promoter. Nuclear extracts were incubated with the ³²P-labelled E-pal probe in the presence of 500-fold molar excess of wild-type (lanes 2 and 5) or mutant cold oligonucleotides (lanes 3 and 6). A black arrowhead indicates a retarded complex. The complete sequence of the E-pal probe is shown at the bottom with the position of the mutated nucleotides indicated by asterisks.