Fig. 5. Direct regulation of the BMP-15 and GDF-9 expression by GCNF. (A) Schematic representation of putative DR0s in the mouse BMP-15 and GDF-9 promoters. (B) EMSAs showing the binding of in vitro translated GCNF to multiple DR0 elements in the BMP-15 and GDF-9 promoters. (a) GCNF was incubated with different ³²P-labeled oligonucleotide probes containing the putative DR0 elements. (b) Retarded DNA-protein complexes in EMSAs by anti-GCNF antibodies (pAb). C1, GCNF–DR0 DNA complexes; C2, antibody retarded GCNF–DR0 DNA complexes. (C) Schematic representation of GCNF expression plasmids and luciferase reporter plasmids containing Oct4, BMP-15 or GDF-9 promoters. (D) Western blot analysis showing the expression levels of full-length GCNF protein and GCNF mutant protein (GCNFΔDBD) in transfected CHO cells using anti-HA antibodies. NS, non-specific protein. (E, G) Dose-dependent repression of the (E) Oct4, (F) BMP-15 and (G) GDF-9 expression by GCNF, not GCNFΔDBD, in CHO cells. Promoter activities are presented as means ± SDs of the percentages of the total promoter activities (without pCMV-HA-GCNF plasmid) and represent three independent measurements.