Fig. 7. Direct binding of Snail to the E-box in claudin-7 and occludin promoters. (A) Electrophoretic mobility shift assay for the interaction of Snail with an E-box in the claudin-7 promoter. ³²P-labeled double-stranded oligonucleotides corresponding to the sequence containing one of the E-boxes of the claudin-7 promoter (E4 in Fig. 6) formed a DNA-protein complex with the in vitro translated/HA-tagged mouse Snail (HA-mSnail) (arrowhead in lane 2), but not with the in vitro translated luciferase (control; lane 1). This complex formation was not observed when mutated oligonucleotides were used (lane 3). This complex formation was affected by an increased amount of unlabeled wild-type oligonucleotides (arrowhead in lanes 4-6), but not by that of unlabeled mutated oligonucleotides (arrowhead in lanes 7-9). The DNA-protein complex band shifted upwards when incubated with anti-HA pAb (arrow in lane 11) but not when incubated with anti-GFP pAb (control, lane 10). (B) Electrophoretic mobility shift assay for the interaction of Snail with an E-box in the occludin promoter. Results were very similar to those shown in A. (C) Biotinylated oligonucleotide precipitation assay. The nuclear extract was prepared from 293 cells transfected with an HA-Snail expression vector (HA-Snail) or an empty vector (con). This extract was incubated with biotin-labeled double-stranded wild-type (wt) or mutated (mut) oligonucleotides corresponding to the sequence containing the E-box of the claudin-7 promoter (E4 in Fig. 6). The oligonucleotides were then recovered using streptavidin-conjugated agarose beads; bound HA-Snail was detected by immunoblotting with anti-HA mAb. HA-Snail bound specifically to the wild type, not mutated, oligonucleotides (lanes 1-3). A similar specific binding was detected when the oligonucleotides of the E-box sequence in the occludin promoter were used (lanes 4-6).