Fig. 6. Basal and inducible activation of GPIX is dependent upon upstream activation of ERK. A, Dami cells were transfected with the GPIX-567 luc reporter and split into two populations. Cells were growth-arrested overnight, and both populations were preincubated with the indicated concentrations of the ERK inhibitor PD98059 or p38 inhibitor SB202190 for 30 min. One population of cells was then incubated with PMA (100 nmol/liter). Luciferase activity was determined 2 days later. {square}, -PMA; {blacksquare}, +PMA. B, after overnight growth arrest, Dami cells were incubated with PD98059 (30 µM) or SB202190 (500 nmol/liter) for 30 min before PMA (100 nmol/liter) treatment. Total RNA was harvested at 12 h, and GPIX mRNA expression was analyzed by Northern blot. Membranes were rehybridized with [32P] GAPDH cDNA to demonstrate the amount of RNA loaded. C, UT-7 EPO/Mpl cells were transfected with the GPIX reporter gene construct GPIX-567 luc. After overnight growth arrest, cells were treated with the above inhibitors at the indicated concentrations for 4 h before TPO exposure. Luciferase activity was determined 2 days later as outlined in "Materials and Methods." {square}, culture in basal culture medium; {blacksquare}, culture in the presence of 20 ng/ml TPO. All of the transfection results represent normalized mean ± SD and are representative of three independent experiments performed in duplicate.