Figure 9.

A: Paxillin is down-regulated in EP₂AS clones. Total cell lysates (20 μg/lane) from two vector controls (M3, M5) and four EP₂AS clones (AS8 and AS9, left; AS3 and AS4, right) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After transfer to nitrocellulose membranes, paxillin immunostaining was performed. The paxillin band is seen at ∼65 kd. The inset represents Ponceau S staining of the nitrocellulose membrane. B: Pharmacological down-regulation of the EP₂ receptor is associated with a further loss in paxillin expression. Top: An immunoblot for paxillin expression is illustrated for two separate clones expressing the EP₂ anti-sense construct (AS8 and AS9). The clonal populations were treated with vehicle or with the EP₂ receptor agonist, 11-deoxy PGE₁ (1 ng/ml) for 11 days before preparation of total cell lysates. Indomethacin (10 μg/ml) was also added to block endogenous PGE₂ production. Bottom: Membrane preparations were prepared from a duplicate set of cells treated with agonist or vehicle as described above for the top panel. EP₂ receptor expression was assessed by Western blotting using the anti-EP₂ receptor antibody.