Phosphorylation of MEK1/MEK2 and MKK3/MKK6 in human neutrophils stimulated by G-CSF, GM-CSF, or TNF. Cells were stimulated with G-CSF (50 ng/mL), GM-CSF (5 ng/mL), or TNF (100 U/mL) for 5 minutes at 37°C. Phosphorylation of MEK1/MEK2 and MKK3/MKK6 was analyzed by immunoblotting using antibody against phosphorylated form of each protein. The cell lysates equivalent to 3 × 10⁶cells were loaded onto each lane. The results shown are representative of three independent experiments.