Dominant-negative Ras N17Ras and GAP have no effect on the EPO-induced activation of ELK. (A) Ten micrograms N17Ras or GAP or empty vector along with 2.5 μg Gal4-Elk fusion protein expression plasmid and luciferase reporter plasmid containing Gal4 binding site were cotransfected into BaF3-EpoR cells. Cells were incubated in the absence of EPO for 14 hours. Then 5 U EPO/mL was added, and the incubation was continued for an additional 5 hours. Cells were harvested for luciferase assay. (B) Ten micrograms activated H-Ras or vector along with 2.5 μg Gal4-Elk fusion protein expression plasmid and luciferase reporter plasmid were cotransfected into BaF3-EpoR cells. Cells were incubated in the presence of EPO for 14 hours and then harvested for luciferase assay. (C) Ten micrograms activated H-Ras along with 10 μg N17Ras or GAP or empty vector and 2.5 μg Gal4-Elk fusion protein expression plasmid and luciferase reporter plasmid were cotransfected into BaF3-EpoR cells. Cells were incubated in the presence of EPO for 14 hours and then harvested for luciferase assay. (D) Ten micrograms dominant-negative Raf-1 S338A, S339A, or empty pCMV-tag2A vector along with 2.5 μg Gal4-Elk fusion protein expression plasmid and luciferase reporter plasmid containing Gal4 binding site were cotransfected into BaF3-EpoR cells. Cells were incubated in serum-free medium for 14 hours. After 25 U EPO/mL was added, the incubation was continued for another 5 hours. Cells were harvested for luciferase assay. (E) Ten micrograms N17Ras or dominant-negative Raf-1 K375M or empty pCMV-tag2A vector along with 2.5 μg Gal4-Elk fusion protein expression plasmid and luciferase reporter plasmid containing Gal4 binding site were cotransfected into Rauscher murine erythroleukemia cells. Cells were incubated in serum-free medium for 14 hours. After 25 U EPO/mL was added, the incubation was continued for another 5 hours. Cells were harvested for luciferase assay. Error bars represent means ± SD.