FIGURE 4.

GATA-1 blocks the Ras/Raf/MEK/ERK pathway through its direct interaction with MEK1. A, NIH3T3 cells (2 × 10⁵ cells seeded in 60-mm dish) were transfected with the indicated expression vectors and the reporter gene (3 × AP-1-Luc) together with pRL-CMV. After 12 h, the cells were serum-deprived for 24 h, then lysed, and subjected to the measurement of the firefly and Renilla luciferase activities. The relative firefly luciferase activities normalized by the Renilla luciferase activities are shown as means ± S.D. of three separate experiments. B, Ba/F3 cells (2 × 10⁶ cells) were transfected with the same vectors as Fig. 4A using Amaxa Nucleofector technology. After 24 h of culture, the cells were lysed and subjected to the measurement of the luciferase activities. C, Ba/F3/N-RasE12/G1ERT cells cultured in RPMI supplemented with 1% FBS were treated with 1 μm 4-HT or vehicle. Total cellular lysates were prepared at the indicated time and subjected to immunoblotting with the indicated Abs. The filters were reprobed with corresponding Abs to confirm that the equal amounts of the proteins were loaded. D, coimmunoprecipitation analyses were performed using 293T cells transfected with HA-tagged GATA-1 and/or Flag-tagged MEK1 as indicated. IP, immunoprecipitation; IB, immunoblotting; α, anti. E, total cellular lysate was prepared from murine BM CD71⁺ cells. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. F. The in vitro binding between GATA-1 and MEK1 was examined by GST pull-down assays. ³⁵S-labeled GATA-1 was incubated with GST-MEK1 bound to glutathione-Sepharose beads, and the binding complex was separated by gel electrophoresis and subjected to autoradiography.