Figure 3.

The human MBN proximal promoter region harbors PU.1, C/EBP, and c-Myb binding sites critical for G-CSF-induced MBN expression in Ba/F3/G-CSFR cells. (A) Wild-type (−658), 5′-deletion mutant (−91), and point mutation constructs (PU.1 mut, C/EBP mut, and Myb mut) of the MBN promoter were fused to the chloramphenicol acetyltransferase (CAT) gene. (B) MBN promoter activity in Ba/F3/G-CSFR cells exposed to G-CSF or IL-3. Indicated MBN promoter constructs (20 μg each) were transfected into Ba/F3/G-CSFR cells, which then were cultured in IL-3- or G-CSF-containing medium. Transfection efficiencies were normalized by cotransfection of a cytomegalovirus–luciferase vector. Cells were harvested 40 h posttransfection, and reporter activity was measured. Open and filled bars represent IL-3- and G-CSF-treated cells, respectively. Error bars indicate the SD from the results of at least three independent experiments. Adjacent Table represents calculated G-CSF/IL-3 activity ratios as well as standard errors (SE) for each construct. CAT activities presented for each MBN promoter construct were calculated relative to the −658 in IL-3 culture conditions.