Functional interaction, GATA-1–PU.1, and GATA-1–c-Myb.

(A) GATA-1, PU.1, and c-Myb activities in each transfectant. Twenty micrograms of functional and nonfunctional reporter gene for GATA-1 (3× WT Mα-Luc and 3× MT Mα-Luc), PU.1 (3× WT-MHC-Luc and 3× MT-MHC-Luc), c-Myb (4× WT-MBS-Luc and 4× MT-MBS-Luc), or a backbone reporter gene (JunB-MP-Luc) was cotransfected with 3 μg pRL-CMV-Rluc by electroporation. After 36 hours, the cells were subjected to the measurement of the firefly and the renilla luciferase activities. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to therenilla luciferase activities. Results are shown as the mean ± SD of triplicate experiments. (B) Reciprocal inhibition, GATA-1–PU.1, and GATA-1–c-Myb in NIH3T3 cells. NIH3T3 cells were cotransfected with various amounts of expression vectors, 1.0 μg of an appropriate reporter gene, and 10 ng of pRL-CMV-Rluc by the calcium phosphate coprecipitation method. After 36 hours of culture, cells were subjected to luciferase assay.