Figure 1.

Dax1 and LRH-1 cooperate to up-regulate Oct4 in mES cells. A, Dax1 and LRH-1 interact in mES cells. mES cell lysates were immunoprecipitated (IP) with anti-LRH-1 antibody and Protein A agarose beads. Bound proteins were subjected to SDS-PAGE, and immunoblotting (IB) was performed with anti-Dax1 antibody as described in Materials and Methods. B, Dax1 up-regulates LRH-1-mediated Oct4-Luc activation in F9 cells. F9 embryonal carcinoma cells in 24-well plates were transfected with 200 ng Oct4-Luc and cotransfected with 200 ng empty vector, pcDNA3.1 LRH-1, and/or empty vector or 150–300 ng Dax1. Luciferase assays were carried out on lysates 48 h after transfection, and values were normalized to Renilla luciferase internal control. Data are presented as relative to empty vector control. *, P < 0.03; **, P < 0.002. C, Overexpression or knockdown of Dax1 in mES cells alters Oct4-Luc reporter expression. mES cells in 24-well plates were transfected with 200 ng Oct4-Luc and cotransfected with 200 ng empty vector, pcDNA3 Dax1, scramble, or short hairpin Dax 1 (shDax1) vector. For Dax1 and empty vector transfection, cells were harvested 48 h after transfection, and lysates were subjected to luciferase assay. shDax1 and scramble transfected cells were harvested 24 h after transfection, and the luciferase assay was carried out as described above. Data are presented as relative to empty vector and scramble controls.