Effects of IL-7 stimulation and IL-7 receptor expression in B cells. (A-B) Whole bone marrow was harvested from TC-PTP+/+ (WT) and TC-PTP−/− (KO) mice at P7 (7 WT, 3 KO). Pre-B cells, generated in vitro as described in “Materials and methods,” were labeled with CFDA-SE for 5 days and stained for surface expression of B220 for flow cytometry. Analysis was gated on B220+ cells to include only B cells. Purity and yield are described in Figure S3. (A) Relative cell number (RCN) is plotted against CFDA-SE fluorescence. The number of cell divisions is indicated next to the corresponding CFDA-SE peak. (B) The mitotic index of TC-PTP+/+ (□) and TC-PTP−/− (▪) B cells is reported as mean ± SD. *P < .001. (C-D) Whole bone marrow was harvested from TC-PTP+/+ (WT) and TC-PTP−/− (KO) mice at P7 (4 WT, 4 KO). Pre-B cells were cultured in vitro, as described in “Materials and methods,” and purity and yield are described in Figure S3. Pre-B cells were harvested and starved prior to stimulation with IL-7 (+IL-7) for 5 minutes or left untreated (unstim). Cell lysates were prepared and fractionated on 8% SDS-PAGE. (C) Western blot analysis of phosphorylated Jak1 (pJak1), total Jak1 (Jak1), phosphorylated Stat5 (pStat5), total Stat5 (Stat5), and TC-PTP was performed. Each sample contained 17 μg protein. (D) Quantitation of Western blot analysis. □, TC-PTP+/+; ▪, TC-PTP−/−. Fold increase in the density of pJak1 and pStat5 are provided relative to unstimulated controls and after normalization to total density of Jak1 and Stat5 protein, respectively. Data are provided as mean ± SD. *P < .01. (E) Whole bone marrow was harvested from TC-PTP+/+ and TC-PTP−/− mice at P7. Ex vivo cells (1 × 106) were stained for surface expression of B220, CD25 and CD43, and CD127 (IL-7Rα) and analyzed by flow cytometry. Analysis was gated on B220+, CD43+ or CD43−, and CD25+ cells to include only pre-B cells. RCN is plotted against CD127 fluorescence. The percentage of CD127− and CD127+ cells is indicated. All experiments were repeated at least 3 times.
