Figure 6.

Time course of the PGE₂-stimulated phosphorylation of the p65 subunit of NF-κB (A) and effects of the PKA inhibitors, H89 and PKI on the PGE₂-stimulated phosphorylation of p65 (B) and on PGE₂-stimulated NF-κB responsive luciferase reporter gene activity (C) in HEK cells stably transfected with the human EP₁ receptor (HEK-EP1). (A) HEK-EP1 cells were treated with 1 µM PGE₂ for indicated times at 37°C and whole cell lysates were prepared and subjected to immunoblot analysis with antibodies against either phospho-p65 or total p65. (B) HEK-EP1 cells were pretreated with vehicle (control) or 10 µM H89 or 5 µM PKI for 30 min 37°C and were then treated with either vehicle (veh) or 1 µM PGE₂ for 3 h. Lysates were prepared and subjected to immunoblot analysis as in panel A. Shown are representative immunoblots that were repeated at least three times for each antibody and condition. (C) HEK-EP1 cells were transiently transfected with a NF-κB luciferase reporter plasmid and ∼18 h later were pretreated for 30 min 37°C with either vehicle (control) or 10 µM H89 followed by treatment with either vehicle (veh) or 1 µM PGE₂. Luciferase activity was determined the next day. Data are the means ± SEM of triplicate measurements from a representative experiment that was repeated three times. ***P < 0.001; one-way anova, followed by Bonferroni post-test.