Phosphorylation of ERK1, ERK2, and p38 MAPK 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 10 minutes at 37°C. Phosphorylation of ERK1, ERK2, and p38 MAPK was analyzed by immunoblotting using antibody against phosphorylated form of each protein (upper panel). The equal loading of proteins onto each lane was confirmed by immunoblotting using antibody that recognizes both phosphorylated and unphosphorylated forms of ERK1/ERK2 or p38 MAPK (lower panel). The cell lysates equivalent to 3.8 × 106cells were loaded onto each lane. The results shown are representative of seven independent experiments. In this experiment, the exposure time was somewhat prolonged to determine whether G-CSF was able to phosphorylate p38 MAPK, which is responsible for higher baseline level of p38 MAPK phosphorylation as compared with that shown in Figs 3, 4, and 7.
