Figure 8

(A,) Electrophoretic mobility shift assays show specific Lef1 binding to 3' sox10 intron 1 DNA sequences contained in Fragment (Frag) B. A specific band of reduced electrophoretic mobility (arrowhead) is observed when the 3' intron 1 fragment B is incubated with in vitro made myc-tagged Lef1 (Lef1^MT) protein but not when incubated with in vitro made GFP protein. The specific Lef1^MT/DNA complex is lost in the presence of anti-myc antibody and replaced by a supershifted band (*); this band is specific to Lef1^MT since this supershift is not seen with an unrelated (anti-WT1) antibody. (B) Competition with excess unlabeled oligonucleotide containing a wild type Lef1 binding site (WT oligo) abolishes the specific binding of Lef1 with Fragment B but oligonucleotide in which the Lef1-binding motif is mutated (mut oligo) is unable to compete for specific binding. (C) Site directed mutagenesis of the conserved Lef1 binding site B1 has little effect on Lef1 binding to Fragment B, additional mutation of site B2 (to give Frag B1,B2) reduces binding of Lef1. (D) Lef1 binding to fragment 2–5 (2–5^WT) is reduced by mutagenesis of sites B1, B2 and site 5 in 2–5^mut. (E). Reduced Lef1 binding to fragment 3–5 containing only two conserved Lef1 binding sequences.