Figure 1

Generation of mice carrying Nkx2-1 mutant alleles. (A) The structure of the Nkx2-1 mutants is schematically shown. Numbering of amino acids is shown according to [19]. P indicates phosphorylated serine residues according to [8]; AD1 and AD2, activation domains. (B) Genomic structure of the Nkx2-1 locus, wild type allele and alleles modified by homologous recombination. Black boxes represent exons; hatched box the homeobox; ATG and TGA codons are indicated. The probe used for genotyping ES cell clones and mice is indicated by a black bar labeled pr. PGKneo, selection marker; pA, SV40 poly(A) sequence; B, BamHI. (C) Southern blot analysis of genomic DNA from mouse tails digested with BamHI and probed probe within indicated in panel B. The lower band corresponds to the mutated allele (4.5 kb), the upper band to the wild type allele (12 kb). (D) Cellular extract from wild type and mutated mouse thyroids (left) were used in EMSA assays with an oligonucleotide containing a high affinity Nkx2-1 binding site. Extracts from FRTL-5 cells transfected with plasmids encoding mutated forms of Nkx2-1 were used as controls (right). Genotype of the mice and plasmids used in trasfected cells are indicated on each lane. (E) Lung homogenates (35 μg of protein) from wild type and PM/PM mice (E18.5) were phosphatase treated (+) or untreated (-), subjected to SDS PAGE, electrophoretically transferred to nitrocellulose and probed with anti Nkx2-1 antibody. The phosphate treatment increases the apparent mobility of wild type Nkx2-1 but does not affect the mobility of PM protein.