Gfi1b’s effect on Rag transcription is mediated in part through Foxo1. (A) Anti-Foxo1 Western blot analysis of whole cell extracts from RAG1-GFP^high cell lines infected with a Gfi1b-ER retroviral overexpression construct and treated (+) or not (−) with tamoxifen (4-OHT) for 12 h. Tubulin was used as a loading control. Data are representative of three independent experiments. (B) Quantitative RT- PCR assay for Foxo1 transcripts in RNA from Gfi1b-ER–overexpressing RAG1-GFP^high cells treated or not (−) with tamoxifen (4-OHT) for the indicated times. Error bars denote standard deviation of triplicate assays. (C, left) Anti-Foxo1 Western blot using whole cell extracts from the indicated AMuLV cell lines and anti-tubulin as a loading control. (C, right) Quantitation of this Western blot showing the ratio of Foxo1 to tubulin signals. Data are representative of three independent experiments. (D) Flow cytometric analysis of RAG1-GFP expression in RAG1-GFP^high parental cells, or RAG1-GFP^high cells overexpressing Gfi1b alone, or Gfi1b and Foxo1. Data are representative of two independent experiments. (E) α-FLAG ChIP-Chip analysis of RAG1-GFP^high and Gfi1b^−/− cells overexpressing FLAG-tagged Gfi1b analyzed using a custom genomic tiling array. Data are shown for the Foxo1 gene locus. Arrows indicate location of primer sets used in F. (F) Quantitative PCR analysis of the indicated ChIP samples conducted on Gfi1b^−/− cells overexpressing FLAG-tagged Gfi1b or on untransduced Gfi1b^−/− cells using either the 1.74B or control primer sets. Error bar represents standard deviation of triplicate PCR assays. Data are representative of two independent experiments.