American Society of Hematology

Fig. 2.

$Fig. 2. Characterization of Py origin-dependent replication by activation of STAT5. / Plasmids containing 4 tandem repeats of STAT5 binding sites fused to Py virus replication origin (A) or luciferase (B) were transfected to BA/F-wild cells. Cells were restimulated with hGM-CSF (10 ng/mL) and harvested after 24 hours of incubation. The tyrosine kinase inhibitor genistein (10 or 20 μg/mL) was added 30 minutes before restimulation. Either DpnI (A) or luciferase assay (B) was done as described in “Materials and methods.” Arrow in (A) indicates the replicated plasmid. (C) Subcellular translocation of STAT5 by hGM-CSF in the presence or absence of hGM-CSF. Cells were stimulated with hGM-CSF (10 ng/mL) in the presence or absence of genistein (20 μg/mL) and separated into soluble and chromatin fractions. Recovery of STAT5 in these fractions was analyzed by Western blotting with anti-STAT5 or phosphotyrosine antibodies.$

Characterization of Py origin-dependent replication by activation of STAT5.

Plasmids containing 4 tandem repeats of STAT5 binding sites fused to Py virus replication origin (A) or luciferase (B) were transfected to BA/F-wild cells. Cells were restimulated with hGM-CSF (10 ng/mL) and harvested after 24 hours of incubation. The tyrosine kinase inhibitor genistein (10 or 20 μg/mL) was added 30 minutes before restimulation. Either DpnI (A) or luciferase assay (B) was done as described in “Materials and methods.” Arrow in (A) indicates the replicated plasmid. (C) Subcellular translocation of STAT5 by hGM-CSF in the presence or absence of hGM-CSF. Cells were stimulated with hGM-CSF (10 ng/mL) in the presence or absence of genistein (20 μg/mL) and separated into soluble and chromatin fractions. Recovery of STAT5 in these fractions was analyzed by Western blotting with anti-STAT5 or phosphotyrosine antibodies.

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