Endothelin Receptor Expression in Skin Samples

Two 3mm³ ventral forearm skin samples were obtained under local anesthesia (2% lidocaine without epinephrine) as previously described(38 (link)). Samples were immediately frozen in liquid nitrogen and stored at -80°C until analysis. The expression levels of ET_AR and ET_BR were determined by Western blotting. Samples were lysed in ice-cold 1× radioimmunoprecipitation assay buffer (Upstate) containing protease inhibitors (Roche), and total protein concentration was determined (Bio-Rad protein assay reagent). Equal amounts of lysate proteins (25 μg) from each sample were resolved by Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose. Blots were blocked with 3% nonfat dry milk for 1 hour at room temperature. The membrane was incubated with primary antibody followed by horseradish peroxidase conjugated anti rabbit or anti mouse antibody (1:1 000) for 1 hour at room temperature. Actin was used as loading control. Mouse monoclonal anti-ET_AR (1:1 000, Abcam), mouse monoclonal anti-ET_BR (1:1 000; Abcam), and mouse monoclonal anti actin (1:5 000; Santa Cruz Biotech) were used. Blots were developed using enhanced chemiluminescence using a ChemiDoc Touch Imaging system (Bio-Rad) and quantified using ImageJ (National Institutes of Health).