Figure 5.

Dax1 regulation of Oct4 is mediated by SRA. A, mES cells were transfected in three 10-cm dishes with 10 μg pDax-1-Myc or pCMV-3tag-4A vector per dish, and RNA-immunoprecipitation was performed as described in Materials and Methods. The Final RNA immunoprecipitate (IP) was used to synthesize cDNA and QPCR was performed for SRA and glyceraldehyde-3-phosphate dehydrogenase (as a nonspecific normalization control). *, P < 0.02. B, mES cells stably expressing shRNA against SRA or scrambled were generated as described in Materials and Methods. RNA was harvested, cDNA was synthesized, and QPCR was carried out to analyze the amount of SRA knocked down. Results are presented as relative to scramble control. C, Scramble or SRA knockdown (KD) mES cells were transfected with 200 ng Oct4-Luc reporter and cotransfected with empty vector or 300 ng pcDNA3 Dax1. Cells were harvested 48 h after transfection and lysates were analyzed for luciferase activity. Reporter alone for each cell line was set as 1 luciferase unit. **, P < 0.006. D, Scramble or SRA KD mES cells were transfected in six-well plates with 3 μg empty vector or Dax1. Cells were harvested 48 h after transfection for RNA, cDNA was synthesized, and QPCR was carried out. Change in Oct4 was normalized, and results are presented as fold change over empty vector control.