American Society of Hematology

Figure 6.

$Figure 6. TPO enhances binding of USF-1 to the HOXB4 promoter. (A) UT-7/TPO cells were stimulated with 100 ng/mL TPO for the indicated times and nuclear fractions were prepared for electrophoretic mobility shift assay (EMSA). EMSA was performed using oligonucleotides corresponding to the putative USF-1–binding site of the human HOXB4 gene promoter. Arrows indicate the DNA-protein complexes. (B) To confirm DNA-binding specificity, a 150 M excess of unlabeled probe was added to the reaction mixture (lane 2). Then, 10 μg of the nuclear extracts was incubated for 20 minutes at 20°C with antibody against USF-1 (lane 4), USF-2 (lane 5), or USF-1 and USF-2 (lane 6). White arrows indicate the shifted complexes. (C) UT-7/TPO cells were pretreated with the indicated concentration of SB203580 for 1 hour and then stimulated with 100 ng/mL TPO for 3 hours. Nuclear proteins were prepared and USF-1–binding activity was assessed by EMSA. (D) Parental UT-7/TPO cells and clone 1 p38-DN MAPK-expressing cells were stimulated with 100 ng/mL TPO for 3 hours and nuclear proteins were prepared for EMSA.$

TPO enhances binding of USF-1 to theHOXB4promoter. (A) UT-7/TPO cells were stimulated with 100 ng/mL TPO for the indicated times and nuclear fractions were prepared for electrophoretic mobility shift assay (EMSA). EMSA was performed using oligonucleotides corresponding to the putative USF-1–binding site of the human HOXB4 gene promoter. Arrows indicate the DNA-protein complexes. (B) To confirm DNA-binding specificity, a 150 M excess of unlabeled probe was added to the reaction mixture (lane 2). Then, 10 μg of the nuclear extracts was incubated for 20 minutes at 20°C with antibody against USF-1 (lane 4), USF-2 (lane 5), or USF-1 and USF-2 (lane 6). White arrows indicate the shifted complexes. (C) UT-7/TPO cells were pretreated with the indicated concentration of SB203580 for 1 hour and then stimulated with 100 ng/mL TPO for 3 hours. Nuclear proteins were prepared and USF-1–binding activity was assessed by EMSA. (D) Parental UT-7/TPO cells and clone 1 p38-DN MAPK-expressing cells were stimulated with 100 ng/mL TPO for 3 hours and nuclear proteins were prepared for EMSA.

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