Gene targeting and manipulation of Foxh1 EH1 domain. (A) The locations of FLAG tag (green), forkhead DNA-binding domain (gray), EH1 motif (red), and Smad-interacting domain (blue) in wild-type and variant Foxh1 proteins. Numbers denote amino acid position. Altered EH1 amino acids in Foxh1mEH1 are in red. (B) Southern blot analysis showing correct targeting of Foxh1 locus in three independent mESC clonal cell lines heterozygous for Foxh1LCA allele. (C) PCR analysis of offspring derived from Foxh1mEH1 chimeras detects two bands, 428 bp and 374 bp, for the Foxh1mEH1 and wild-type Foxh1 alleles, respectively.