Effect of U0126 on ROS production and ERK1/2 phosphorylation in neutrophils from healthy volunteers.

(A) Isolated neutrophils were preincubated with 10 μM U0126 for 30 minutes and stimulated for 20 minutes with 1 μM fMLP with or without priming with 5 ng/mL GM-CSF. ROS production was measured by FACS analysis. Results represent the mean increase in ROS produced after stimulation, compared with unstimulated cells, of 4 individual experiments. Significant reduction of the respiratory burst by U0126 is indicated by an asterisk (P < .05). (B) Neutrophils were stimulated with 1 μM fMLP with or without priming with 5 ng/mL GM-CSF or with GM-CSF alone. Phosphorylation of ERK1/2 was detected by Western blot analysis and immunodetection. To confirm equal loading, the total amount of ERK1/2 was detected using the ERK1/2 K23 antibody (lower panel). A representative blot is shown of 3 independent experiments. (C) Neutrophils were preincubated with 10 μM U0126 for 30 minutes when indicated and were stimulated for the indicated amount of time with 1 μM fMLP, with or without priming with 5 ng/mL GM-CSF or with GM-CSF alone. Cell lysates were analyzed by Western blotting, using an antibody against phosphorylated ERK1/2 (upper panel). Equal protein loading in the samples was confirmed by immunodetection of ERK1/2 (lower panel).