Mutation of hepatocyte nuclear factor–1β inhibits Pkhd1 gene expression and produces renal cysts in mice
J. Clin. Invest. Thomas Hiesberger, et al. 113:814 doi:10.1172/JCI20083 [Go to this article.]

Figure 4
Effects of DN-HNF1β on DNA binding and promoter activity. (A) EMSA performed using a 44-bp DNA fragment containing the consensus HNF-1 site and nuclear extracts from HeLa cells transfected with expression plasmids encoding V5-tagged HNF-1β (lanes 1 and 2), E12 (lanes 3 and 4), or DN-HNF1β (lanes 5 and 6). Binding reactions were performed in the presence of anti-V5 Ab (lanes 2, 4, and 6) or no Ab (lanes 1, 3, and 5). Arrow indicates retarded band, and arrowhead indicates supershifted band. (B) HEK293 cells were transfected with plasmids encoding FLAG-tagged HNF-1β (lanes 1–3), V5-tagged HNF-1β (lanes 1, 3, 4, and 6), or FLAG-tagged DN-HNF1β (lanes 4–6). Cell lysates were immunoprecipitated (IP) with anti-V5 Ab (lanes 1, 2, 4, and 5) or anti-Myc Ab (lanes 3 and 6), and the immune complexes were subjected to immunoblot analysis with HRP-conjugated anti-FLAG Ab. (C) EMSA performed with the 44-bp DNA fragment and reticulocyte lysates programmed with the indicated amounts (nanograms) of plasmids encoding V5-tagged HNF-1β or FLAG-tagged DN-HNF1β. Binding reactions were performed in the presence of anti-V5 Ab (lanes 6–9), anti-FLAG Ab (lanes 10–13), or no Ab (lanes 1–5). Arrow indicates retarded band, and arrowhead indicates supershifted band. (D) Luciferase activity in mIMCD-3 cells cotransfected with a reporter plasmid containing the 444-bp Pkhd1 promoter and the indicated amounts (nanograms) of an expression plasmid encoding DN-HNF1β. Data presented are mean ± SE of nine independent transfections. *P < 0.05 compared with cells transfected with empty expression plasmid.