HA-tagged Snail was expressed in ATDC5 cells and the nuclear extract was incubated with double-stranded radiolabeled 24-mer oligonucleotide probes containing E-box4 (lane 2–6).
Shifted bands (arrow and dotted) were detected when the probes were incubated with the nuclear extract (lane 2) but not without the nuclear extract (lane 1). For supershift analysis, anti-HA antibody (lane. 3) or non-specific IgG (lane 4) was added. A supershifted band (blanket) was detected only when the probes were incubated with anti-HA antibody. For competition analysis, a 50-fold molar excess of unlabeled wild type probe (lane 5) or 50-fold molar excess of unlabeled mutated probe was added (lane 6). The shifted band disappeared when the probes were incubated with wild type probe (lane 5), but not when incubated with mutated probes. The non-specific band is marked with a dot.