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Fig. 4. Distinct metabolic pathways can lead to the upregulation of neutrophil COX-2: implication of PI-3K, ERK1/2, p38 MAPK and cAMP pathways. (A) PMNs were stimulated in adenosine-free conditions (i.e. in the presence of ADA) with fMLP (upper) or LPS (lower), alone or in combination with the indicated metabolic inhibitors. Samples were processed for the determination of COX-2 by western immunoblotting. (B) PMNs were incubated in adenosine-free conditions (i.e. in the presence of ADA) with a cell-permeable stable analog of cAMP, Sp-cAMPs-am (50 µM), alone or in combination with indicated metabolic inhibitors. Samples were processed for the determination of COX-2 and phosphorylated (P-)CREB by western immunoblotting. In each panel, one typical immunoblot is shown and is representative of at least n=3 independent experiments performed in identical conditions with different donors. Bar graphs depict the expression of COX-2 protein levels, relative to those observed in A: PMNs stimulated with fMLP or LPS only (solid black bars) and in B: PMNs incubated with Sp-cAMPs-am only (solid black bar) as determined by densitometric analysis of the bands in three independent experiments (mean±s.e.m.; n=3). *Statistically different from diluent-treated cells. H89 and KT 5720 (two structurally distinct inhibitors of PKA; 10 µM and 3 µM, respectively), PD 98059 (MEK-ERK1/2 inhibitor; 10 µM), U0126 (ERK1/2 inhibitor; 10 µM), wortmannin (PI-3K inhibitor; 200 nM), SB 203580 (p38 inhibitor; 10 µM).
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