Figure 5.

Effect of PGE₂ and EP receptors on p47phox phosphorylation during K. pneumoniae infection. (A) Rat AMs (4 × 10⁶/well) were pretreated for 20 minutes with the EP2 antagonist, AH-6809 (100 μM), the EP4 antagonist, Ono-AE3-208 (1 μM), or vehicle, or exposed for 5 minutes to PGE₂ (1 μM) before the addition of 50:1 opsonized K. pneumoniae for an additional 5 minutes. Cells were then harvested and fractionated as described in Materials and Methods. The cytosolic fraction was subjected to immunoprecipitation for p47phox, followed by immunoblot analysis using an anti-phosphoserine antibody (1:1,000). Subsequently, the membrane was stripped and probed for total p47phox (1:500). Results are representative of three experiments. (B) Relative phosphorylation was determined by densitometric analysis of immunoblots from three different experiments and expressed as percent of control. *P < 0.05 versus control; ^#P < 0.05 versus K. pneumoniae group by ANOVA.