Effect of PD98059 on tyrosine phosphorylation of ERK1, ERK2, and p38 MAPK in human neutrophils stimulated by G-CSF, GM-CSF, or TNF. Cells were pretreated with PD98059 (100 μmol/L) for 30 minutes at 37°C and thereafter stimulated with G-CSF (50 ng/mL), GM-CSF (5 ng/mL), or TNF (100 U/mL) for 10 minutes at 37°C. Phosphorylation of ERK1, ERK2, and p38 MAPK was analyzed by immunoblotting using antibody against phosphorylated form of each protein. The cell lysates equivalent to 2.8 × 10⁶ cells were loaded onto each lane. The results shown are representative of three independent experiments.