Fig. 5. Identification of the DNA sequence motif bound by Six3. (A) The sequence of the 19 oligonucleotides containing a core ATTA (bold) motif selected by the GST-Six3 fusion protein are aligned for comparison. The oligonucleotide at the top was used in the electrophoretic mobility shift assay (EMSA) experiments depicted below. The DNA sequence identified by Kawakami et al. (Kawakami et al., 1996b) as recognized by Six2, Six4 and Six5 is shown at the bottom. (B) Six3 bound specifically to the identified oligonucleotides in an EMSA. Double-stranded DNA of the first oligonucleotide represented in A was end-labeled with 32P and used as a probe. Lane 1, 32P-labeled probe alone; lane 2, GST and 32P-labeled probe; lane 3, when the GST-Six3 fusion protein was combined with the 32P-labeled probe, specific retardation was observed (bottom arrow); lane 4, a super-shift (top arrow) of the GST-Six3-probe complex was seen when using an anti-Six3 antibody; lane 5, the binding complex was competed when adding 100 times more of the nonradioactive probes than of the radioactive ones; lane 6, competition of the binding complex with 300 times more nonradioactive probes than radioactive probes; lanes 7 and 8, no competition of the binding complex was observed when using either 100 or 300 times more nonradioactive mutated probes, in which the core motif ATTA was mutated into AGCA.
