Fig. 7.

AR represses expression from a multimer of a known Nkx2.1 binding site, and overexpression of Nkx2.1 represses GnRH-P. A, GT1-7 cells were transiently transfected with AR and/or Nkx2.1 expression plasmids and reporter plasmids containing multimers of known Oct, Pbx, or Nkx2.1 binding sites. Cells were treated for 24 h with 100 nm R1881 (closed bars) or ethanol vehicle (open bars) and subjected to luciferase assay. Data were expressed relative to empty vector without AR (data not shown). *, P < 0.05; ***, P < 0.001 vs. vehicle; †, P < 0.001 vs. vehicle-treated 3xNkx2.1 (without Nkx2.1 overexpression) by one-way ANOVA followed by Student's t test. B, GT1-7 cells were transiently transfected with AR and/or Nkx2.1 expression plasmids and GnRH-P. Cells were treated for 24 h with 100 nm R1881 (closed bars) or ethanol vehicle (open bars) and subjected to luciferase assay. Data were expressed relative to vehicle-treated control. Bars with different letters are significantly different from each other by one-way ANOVA followed by Tukey HSD post hoc test. C, Cell lysates from GT1-7 cells, transfected with AR, were immunoprecipitated with an antibody specific for AR (or IgG) followed by immunoblotting with an antibody specific for Prep1 (top) or AR (bottom). IP, Immunoprecipitation; WB, Western blot.