Figure 3. Foxo1 is necessary for inducing two key components of pre-BCR signaling.

IL-7 signaling was attenuated in IRF-4,8^−/− pre-B cells as described in Fig. 1. (a) RT-PCR and immunoblot analysis of Blnk expression. (b) Flow cytometric analysis of intracellular Blnk and Syk 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 Blnk and isotype control antibodies (filled gray histogram) are shown. (c) Immunoblot analysis of indicated proteins in IRF-4,8^−/− pre-B cells transduced with a control or Foxo1 knock down vector encoding a hygromycin resistance gene as a selectable marker. Cells were transduced with retroviral vectors encoding short hairpin (sh) sequences targeting Foxo1 or Luciferase (control) transcripts. Infected cells were sorted based on hygromycin resistance under IL-7 Hi conditions, and plated in IL-7 Lo conditions for 48 h before generating lysates for immunoblotting. (d) Immunoblot analysis of Syk, Blnk, Pax5 and HPRT in IRF-4,8^−/− pre-B cells transduced with a control or Foxo1 retrovirus vector. Cells were maintained in IL-7 Hi conditions and sorted based on GFP expression after transduction as described in Fig. 1f. MigR, MigR-Foxo1 denote control and Foxo1, respectively. (e) Immunoblot analysis of Syk and HPRT upon attenuation of IL-7 signaling in IRF-4,8^−/− pre-B cells (see Fig. 1 for details). (f) Immunoprecipitation of Blnk complexes. Whole cell extracts were prepared from IRF-4,8^−/− pre-B cells cultured in IL-7 Lo conditions for 48 h. Immunoprecipitations were performed using either control IgG or αBlnk antibodies followed by immunoblot analysis with αSyk or αBlnk. 2% of the pre-immunoprecipitated samples were used for input. All data are representative of three independent experiments except for 3c which is representative of two independent experiments.