ET-1 activates caspase-8. Lysates normalized for protein content were analyzed for caspase activation by immunoblot analysis. Un, unstimulated control. (a) ET-1 activates caspase-8 in melanocytes (left panel) and melanoma cells. Asterisks indicate positions of caspase-8 cleavage products. (b) IETD blocks caspase-8 activation by ET-1 in melanoma cells. Identical results were obtained using melanocytes (data not shown). (c) ET-1 does not activate caspase-3 and caspase-7 in melanoma cells. Expected molecular weight of caspase catalytic subunits is approximately 20 kDa. Identical results were obtained using melanocytes (data not shown). (d) Caspase-8 associates with β-catenin, (e) p120CTN, and (f) E-cadherin. (g) Association of β-catenin with E-cadherin in unstimulated versus ET-1–stimulated melanocytes (CL-NHEM) and melanoma cells (SKMEL28). β-catenin immune complexes were prepared from samples normalized for β-catenin content (not total protein content). (h) The 95-kDa E-cadherin fragment is detected in cell culture medium. Lane 1, full-length E-cadherin in whole cell lysates. Lanes 2–5, 95-kDa E-cadherin fragment immunoprecipitated from cell culture medium of cells treated with ET-1 for 40 hours (lanes 3 and 5) and unstimulated controls (lanes 2 and 4). Ab’s directed to a large C-terminal region spanning the caspase cleavage site (ECAD-CT), as well as one directed against the extracellular domain (ECAD-NT), were used. (i) Conditioned medium collected from SKMEL28 cells (SKCM) treated for 40 hours with ET-1 (ET) and unstimulated controls (Un) was transferred to new SKMEL28 cells for the indicated times. Identical results were obtained using melanocytes (data not shown). Results shown are representative of at least four independent experiments. Cas, Caspase.