New NR5A1 mutations and phenotypic variations of gonadal dysgenesis
Fig 4
Electrophoretic mobility shift assay of NR5A1 mutants.
A) DNA-binding capability of mutant NR5A1-T40P and NR5A1-18DKVSG22del were compared to wt-NR5A1 using a SF-1 responsive element of the mouse Amh promoter. Nuclear extracts containing WT-SF1 shifted the labelled probe (arrow). A 200 fold excess of unlabelled competitor abolished the shifted signal, while a 200 fold excess of unlabelled mutated competitor rescues the shifted signal. Both mutant proteins have lost their DNA-binding capability. B) Aliquots of the nuclear extracts used in EMSA were separated by SDS gel electrophoresis, transferred to a nitrocellulose membrane and stained with Ponceau S and anti-myc antibodies to verify equal SF-1 protein concentrations.