Phosphorylation of ERK1, ERK2, and p38 MAPK in human neutrophils stimulated by G-CSF. Cells were stimulated with indicated concentrations of G-CSF for 10 minutes at 37°C or stimulated with 50 ng/mL G-CSF for indicated periods at 37°C. Phosphorylation of ERK1, ERK2 (upper 2 panels), and p38 MAPK (lower panel) was analyzed by immunoblotting using antibody against phosphorylated form of each protein. The cell lysates equivalent to 2.7 × 106 cells were loaded onto each lane. The results shown are representative of five 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.
