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. Author manuscript; available in PMC: 2014 May 20.
Published in final edited form as: Nat Immunol. 2012 Jan 22;13(3):300–307. doi: 10.1038/ni.2210

Figure 1. The IL-7R/PI3K/Akt pathway negatively regulates FoxO activity and Rag gene expression in pre-B cells.

Figure 1

IL-7 signaling was attenuated in IRF-4,8−/− pre-B cells by reducing the concentration of IL-7 in the culture medium from 5.0 ng/mL (IL-7 Hi) to 0.1 ng/mL (IL-7 Lo) for a period of 48 h (a through d). (a) Immunoblot analysis of Foxo1, FoxO3a and FoxO4. Expression of these proteins was normalized to HPRT. (b) Immunoblot analysis of phosphorylated forms of Foxo1 (p-Foxo1), FoxO3a (p-FoxO3a) and Akt (p-Akt) in relation to total Akt (Akt). (c) ChIP analysis of Foxo1 binding to regulatory elements (Erag1,2,3) in the Rag locus1, 3. ChIP was performed with control IgG or αFoxo1 antibodies. Binding enrichment at the indicated Erag region is displayed as a percentage of input DNA and was calculated using quantitative PCR. (d) RT-PCR analysis of Rag1,2 transcripts normalized to those for β-2 microglobulin (β-2mi). For a–d, data are representative of three independent experiments, and error bars show the standard deviation from the mean value. (e) Flow cytometric analysis of IL-7Rα (surface) as well as p-Akt and Foxo1 (intracellular) expression in large cycling and small resting pre-B cells. Bone marrow pre-B cells were isolated from wild type mice (n=3) as detailed in Methods. Flow cytometry profiles of large (red) versus small pre-B cells (green) with the indicated antibodies and an isotype control (filled gray histogram) are shown. Data is representative of analysis of pre-B cells from three individual mice. (f) Effect of pre-BCR expression on Akt activity in pro-B cells. Rag2−/− pro-B cells were transduced with either MigR (Control) or MigR-μH chain (WT383) retroviral vectors and the infected cells were sorted based on expression of GFP. Flow cytometry analysis was used to confirm the surface expression of the pre-BCR (left panel). Phospho- and total Akt levels were monitored by immunoblotting in the indicated cells under IL-7 Hi or Lo conditions as detailed above. Data is representative of two independent experiments.