Effect of LY294002 on the fMLP- and GM-CSF–induced phosphorylation of ERK1/2 in human neutrophils.

Neutrophils from a healthy volunteer were preincubated with the indicated concentrations of LY294002 for 30 minutes. Neutrophils were subsequently stimulated for 2 minutes with 1 μM fMLP alone, fMLP following priming of the neutrophils with 5 ng/mL GM-CSF, or with 5 ng/mL GM-CSF alone. Stimulation was stopped by the addition of 1 × Laemmli buffer to the pelleted neutrophils and by boiling the samples. Proteins were separated by SDS-PAGE, and Western blotting was performed using an antibody against phosphorylated ERK1/2 (upper panel). Lysates of unstimulated cells were run as negative controls (C). Equal loading was demonstrated by reprobing the blots with an antibody against ERK1/2 (lower panels).