Direct interaction of Six and Eya on the DNA. (A) Gel retardation assays of Eya3 and Six proteins. Six nanograms of GST-Six1 (lanes 1 to 3), 300 ng of GST-Six2 (lanes 4 to 6), and 6 ng of GST-Six4 (lanes 7 to 9) were incubated with 5 fmol of the C3 probe. One-and-a-half micrograms of GST-Eya3 (lanes 2, 3, 5, 6, 8, and 9) and 0.2 μl of anti-Eya3 sera (lanes 3, 6, and 9) were added. Arrowheads show positions of Six-DNA probe complex. The Eya-Six-DNA complex is shown by arrows, and the supershifted complex is shown by anti-Eya3 by asterisks. (B) Scheme of deletion mutation of Eya3 protein used in Fig. 1C. Positions of amino acids in Eya3 protein deletions are shown. (C) Fifteen-and-a-half picomoles each of full-length GST-Eya3 (lane 2), GST-Eya3CΔ2 (lane 4), GST-Eya3EF1 (lane 5), GST-Eya3/62aa+EF1 (lane 6), GST-Eya3NΔ2 (lane 7), and GST-Eya3NΔ1 (lane 8), and 2.2 pmol of GST-Eya3CΔ1 (lane 3) were added in the presence of 4 ng of GST-Six4 with the C3 probe. Arrowhead shows the position of the GST-Six4-DNA probe complex.