Figure 3.

Alteration of EP₂ receptor expression and function in clonal populations of HaCat cells stably transfected with EP₂ sense and EP₂AS expression constructs. A and B: Agonist-induced cAMP was measured in stably transfected clonal populations of HaCat cells expressing the EP₂ receptor in sense (A) or anti-sense (B) orientation. EP₂ sense clones (S1 to S6), EP₂AS clones (AS1–6, 8, and 9), and empty vector pMIRB control clones (M1 to M5) were pretreated with indomethacin (10 μg/ml) overnight to block endogenous PGE₂ formation. cAMP was then measured after a 15-minute stimulation with the EP₂ agonist 11d-PGE₁ (1 μg/ml). Isobutyl methylxanthine (2 mmol/L) was included to block cAMP phosphodiesterase activity. C and D: EP₂ receptor expression was evaluated by Western blot in both EP₂ sense clones (18 μg/lane) (C, top) and EP₂AS clones (12 μg/lane) (D, top) compared with two vector control clones (M3 and M5). Immunoblotting was performed using the monoclonal anti-hEP₂ receptor antibody (clone 2B4). C, bottom: Coomassie-stained gel (after transfer). D, bottom: Ponceau S stain of the membrane after transfer.