American Society of Hematology

Figure 4

$Figure 4. Stat1 phosphorylation in bone marrow B cells. (A) Whole bone marrow was harvested from TC-PTP+/+ (□) and TC-PTP−/− (▪) mice at P7. Ex vivo cells were stained for surface expression of B220 and intracellular expression of Stat1 and phosphorylated Stat1 (pStat1), for flow cytometry analysis. Nonspecific IgG was used as negative control for intracellular staining. Analysis was gated on B220+ cells to include only B cells. RCN is plotted against pStat1, Stat1, or IgG fluorescence. MFI is indicated. (B) Whole bone marrow was harvested from TC-PTP+/+ (WT) and TC-PTP−/− (KO) mice at P7 (3 WT, 3 KO). Ex vivo pre-B–cell lysates were prepared and fractionated on 8% SDS-PAGE. Western blot analysis of phosphorylated Stat1 (pStat1), total Stat1 (Stat1), and β-actin was performed in duplicate. Each sample contained 15 μg protein. (C) Whole bone marrow was harvested from TC-PTP+/+ and TC-PTP−/− mice at P7 (4 WT, 3 KO). Pre-B cells were cultured in vitro as described in “Materials and methods”; purity and yield are described in Figure S3. Pre-B cells were harvested and starved prior to stimulation with IFN-γ (+IFN-γ) for 5 minutes or left untreated (unstim). Cell lysates were prepared and fractionated on 8% SDS-PAGE. Western blot analysis of phosphorylated Stat1 (pStat1), total Stat1 (Stat1), TC-PTP, and β-actin was performed. Each sample contained 3.25 μg protein. (D) Quantitation of Western blot analysis. □, TC-PTP+/+; ▪, TC-PTP−/−. (D left panel) Fold increase in the density of pStat1 is provided relative to unstimulated controls and after normalization to density of β-actin (Actin) and total density of Stat1. (D right panel) Relative density of total Stat1 protein before and after normalization to total density of β-actin. Data are provided as mean ± SD. *P < .01. All experiments were repeated at least 3 times. All experiments were repeated at least 3 times.$

Stat1 phosphorylation in bone marrow B cells. (A) Whole bone marrow was harvested from TC-PTP^+/+ (□) and TC-PTP^−/− (▪) mice at P7. Ex vivo cells were stained for surface expression of B220 and intracellular expression of Stat1 and phosphorylated Stat1 (pStat1), for flow cytometry analysis. Nonspecific IgG was used as negative control for intracellular staining. Analysis was gated on B220⁺ cells to include only B cells. RCN is plotted against pStat1, Stat1, or IgG fluorescence. MFI is indicated. (B) Whole bone marrow was harvested from TC-PTP^+/+ (WT) and TC-PTP^−/− (KO) mice at P7 (3 WT, 3 KO). Ex vivo pre-B–cell lysates were prepared and fractionated on 8% SDS-PAGE. Western blot analysis of phosphorylated Stat1 (pStat1), total Stat1 (Stat1), and β-actin was performed in duplicate. Each sample contained 15 μg protein. (C) Whole bone marrow was harvested from TC-PTP^+/+ and TC-PTP^−/− mice at P7 (4 WT, 3 KO). Pre-B cells were cultured in vitro as described in “Materials and methods”; purity and yield are described in Figure S3. Pre-B cells were harvested and starved prior to stimulation with IFN-γ (+IFN-γ) for 5 minutes or left untreated (unstim). Cell lysates were prepared and fractionated on 8% SDS-PAGE. Western blot analysis of phosphorylated Stat1 (pStat1), total Stat1 (Stat1), TC-PTP, and β-actin was performed. Each sample contained 3.25 μg protein. (D) Quantitation of Western blot analysis. □, TC-PTP^+/+; ▪, TC-PTP^−/−. (D left panel) Fold increase in the density of pStat1 is provided relative to unstimulated controls and after normalization to density of β-actin (Actin) and total density of Stat1. (D right panel) Relative density of total Stat1 protein before and after normalization to total density of β-actin. Data are provided as mean ± SD. *P < .01. All experiments were repeated at least 3 times. All experiments were repeated at least 3 times.

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