Fig. S2

Lef1 and Tcf7 associate with foxj1a regulatory sequence. (A) lef1 and tcf7 are both expressed in DFCs (Arrow points to DFCs). (B) Lef1 coprecipitates with foxj1a regulatory sequence by a chromatin-immunoprecipitation assay. Briefly, embryos injected with hsp:lef1-GFP plasmid were heat shocked at 30% epiboly and collected at the bud stage. Anti-GFP antibody was used to immunoprecipitate Lef1-DNA complexes, and the recovered DNA was subjected to polymerase chain reaction analysis using primer pairs amplifying 2 approximately 0.2-kilobase (kb) fragments of the 0.6-kb foxj1a regulatory sequence. One DNA fragment harbors 1 putative Lef/Tcf binding site (D1), and the other fragment contains 2 sites (D2 and D3). A primer pair that amplifies a region more than 2 kb away from the enhancer sequence served as a negative control. (C) Tcf7 binds to the foxj1a regulatory sequence. A Tcf7-GST fusion protein (100 ng) was incubated with ³²P–labeled oligonucleotides corresponding to D1, D2, and D3 sequences. The resulting products were loaded onto a 6% acrylamide non-denaturing gel.