Fig. 2.

Lef1 binds to the foxj1a regulatory sequence. (A) Schematic diagram of a 0.6 kb foxj1a regulatory sequence. This sequence is located approximately −5.2 kb to −4.6 kb upstream of the ATG start codon. D1, D2, and D3 represent putative Lef1/Tcf binding sites. (B) Lef1 interacts with the foxj1a enhancer by electrophoretic mobility shift assay. Lef1-GST fusion protein (100 ng) or glutathione S-transferase (GST) alone (100 ng) was incubated with a ³²P-labeled 0.6-kb foxj1a regulatory sequence in the presence or absence of 50-fold excess of cold probe. The resulting products were loaded onto a 4% acrylamide non-denaturing gel. (C) Lef1 predominantly binds to the D2 site. A Lef1-GST fusion protein (100 ng) was incubated with ³²P-labeled oligonucleotides corresponding to D1, D2, D3, and mutated D2 (D2m) sites in the presence or absence of 100-fold excess of unlabeled oligonucleotide. The reaction mixtures were loaded into a 6% acrylamide non-denaturing gel. TCR indicates T-cell antigen receptor enhancer AAGTTTC motif, serving as a positive control for Lef1 binding.