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Figure 2. IL-10 inhibits GM-CSF-induced ERK1/ERK2 activity without affecting ERK phosphorylation status. A) PMN (25x106/ml) were incubated with buffer alone or with IL-10 (50 ng/ml), GM-CSF (15 ng/ml), or GM-CSF (15 ng/ml) plus IL-10 (50 ng/ml) for various times at 37°C. Western blots were performed with anti-human phospho-p44/42 MAPK antibodies (P-ERK). B)PMN (25x106/ml) were stimulated where indicated with 15 ng/ml GM-CSF in the presence or absence of various concentrations of IL-10. Western blots were performed with anti-human phospho-p44/42 MAPK antibody C) PMN (25x106/ml) were stimulated with 15 ng/ml GM-CSF in the presence or absence of various concentrations of IL-10. Where indicated, PMNs were pretreated with PD98059 (50 µM) for 30 min before stimulation. ERK1/2 was immunoprecipitated, and kinase assay was performed in the presence of MBP and [
32P]ATP. MBP phosphorylation was detected by autoradiography (P-MBP). D) PMN (25x106/ml) were stimulated with 15 ng/ml GM-CSF at 37°C for 20 min in the presence or absence of various concentrations of IL-10. Immunoprecipitation was performed using anti-ERK1/2 antibodies (IP anti-ERK1/2) or an irrelevant isotypic IgG control Ab (IP IgG control). Kinase assay was performed with MBP (top) or recombinant p47PHOX (bottom) as substrate. MBP and p47PHOX phosphorylation were detected by autoradiography (P-MBP and P-p47PHOX respectively).
