Figure 3.

The effects of the NF-κB inhibitor, BAY 11–7082, and dominant negative CREB (DN-CREB) on PGE₂-stimulated Nurr1 protein expression (A) and on PGE₂-stimulated Nurr1 responsive (NBRE) luciferase reporter gene activity (B) in HEK cells stably expressing the human EP₁ receptor. (A) HEK-EP1 cells were transiently transfected with DN-CREB ∼18 h prior to the experiment or were pretreated the day of the experiment with either vehicle (control) or 10 µM BAY 11–7082 for 30 min at 37°C, followed by treatment with either vehicle (veh) or 1 µM PGE₂ for 3 h at 37°C. Lysates were prepared and subjected to immunoblot analysis using antibodies against either Nurr1 or vinculin. A representative immunoblot is shown from one of three independent experiments. (B) HEK-EP1 cells were transiently transfected either alone with a NBRE luciferase reporter plasmid or together with a plasmid encoding DN-CREB as described in the methods section and ∼18 h later were pretreated with either vehicle (control) or 10 µM BAY 11–7082 for 30 min at 37°C, followed by treatment with either vehicle (veh) or 1 µM PGE₂. Luciferase activity was determined the next day. Data are the means ± SEM of quadruplicate measurements from a representative experiment that was repeated three times. ***P < 0.001; one-way anova, followed by Bonferroni post-test.