The greatest increase was observed in the 30-kd band. membrane pellets were isolated as described. 32 Total cell lysates were prepared by scraping washed cells into nonreducing 4% Laemmlis buffer. Protein was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Immunoblots EP2 receptor immunoblots were performed GREM1 using membrane preparations that were boiled for 5 minutes in an equal volume of 4% Laemmlis buffer containing bromphenol blue and 4% -mercaptoethanol. Samples were then electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels using prestained protein molecular weight markers. After transfer to nitrocellulose or polyvinylidene difluoride membranes, Ponceau S staining of membranes or Coomassie staining (GelCode Blue, Pierce Chemicals, Rockford, IL) of the gels was performed to verify equal loading. Blocking was done using Tris-buffered saline containing 0.05 to 0.1% Tween 20, 5% nonfat dry milk, and 1% normal goat serum. Immunodetection was performed using mouse monoclonal anti-human EP2 receptor antibody (clone 2B4) at a concentration of 0.4 g/ml for 1 hour at room temperature followed by sheep anti-mouse IgG-F(ab)2-peroxidase conjugate (1:2000) for 30 to 45 minutes. Primary and secondary antibodies were diluted in TBST containing 1% milk and 1% normal goat serum. Immunoreactive bands were detected by enhanced chemiluminescence (Renaissance ECL reagent; NEN Life Sciences, Boston, MA). Extremely careful and consistent washing and blocking was required with this antibody to avoid numerous nonspecific bands. For peptide competition, anti-EP2 receptor antibody was incubated with or without 0.5 to 1 1 mg of EP2-free peptide dissolved in 1 ml of 100 mmol/L Tris, pH 8.0, containing 0.15 mol/L NaCl. After incubation for 1 to 2 2 hours at 37C, the reaction RGH-5526 products were diluted with TBST containing 1% milk and 1% normal goat serum as above. For paxillin immunostaining, 40 g of total cell lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as above. After incubation with mouse monoclonal anti-paxillin antibody (Transduction Laboratories, Lexington, KY), immunoreactive bands were detected as above. Cyclic-AMP Assays Transfectants were plated at a density of 40,000 cells/well into 48-well plates. One to 2 days after reaching confluence, media was suctioned and replaced with growth media containing 10 g/ml of indomethacin. After 2 to 3 3 hours at 37C, the monolayer was washed twice with PBS containing 10 g/ml of indomethacin. After washing, growth media containing 10 g/ml of indomethacin was added. The cells were then incubated overnight (20 to 22 hours) after which the monolayers were washed four times with serum-free Dulbeccos modified Eagle medium containing 25 mmol/L of HEPES and 10 g/ml of indomethacin (reaction buffer). After washing, 0.25 ml of reaction buffer (prewarmed to 37C) was added and the cells were incubated at 37C for 15 minutes. The EP2 receptor agonist, 11d-PGE1 (2 g/ml), was then added in 0.25 ml of reaction buffer containing 4 mmol/L of 3-isobutyl-1-methylxanthine. Plates were incubated for 15 minutes at 37C after which the media was aspirated and ice-cold 0.1 N of hydrochloric acid (HCl) was added. Levels of cAMP were quantitated in the acid supernatants with a commercial cAMP EIA kit (Assay Designs, Ann Arbor, MI). Protein content in the 0.1 N HCl supernatants was quantitated using the BCA protein assay (Bio-Rad). Skin Equivalents Fibroblast-containing dermal equivalents were first prepared using primary adult human fibroblasts at passage 3 to 5 5 by a modification of a previously described method. 43 Dermal equivalents were prepared using 5 105 primary human fibroblasts and purified rat tail type I collagen (Collaborative Research/Becton Dickinson, BD Biosciences, Bedford, MA). Each dermal equivalent was allowed to contract for 4 to 7 days in a 30-mm tissue-culture dish. Skin RGH-5526 equivalents were then prepared. Clonal populations of HaCat cells overexpressing the EP2 receptor in sense or anti-sense orientation and vector control cells were trypsinized and 1 to 2 2 105 cells were seeded onto the dermal equivalents using a 5-mm internal diameter sterile glass cloning chamber. After 2 to 3 3 hours the cloning chambers were removed, and the skin equivalents were submerged in HaCat growth media. After 7 to 10 days, the skin equivalents were RGH-5526 lifted to the air-liquid interface on sterile wire grids. After 3 to 7 days, the lifted skin equivalents were fixed in 10% buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin (H&E) for light microscopic examination. For butaprost- and 11d-PGE1-treated clones (see Figures 7 and 8 ? ? ), cells were pretreated with agonist for 3 days before seeding onto dermal equivalents. Growth media containing butaprost, 11d-PGE1, or vehicle was changed every 2 days during both submerged culture and culture at the air-liquid interface. Open in a separate window Figure 7. EP2 agonist exposure results in a marked increase in invasiveness in vector control clones. A: Three vector control clones were.