Analysis of the agonist's site of action, using characterized Hh-pathway antagonists. (a) The Hh-signaling pathway. The major components are shown, along with the suspected sites of action of four antagonists: 5E1, the Hh-ligand-binding/blocking monoclonal antibody; cyclopamine, the natural product inhibitor, activity of which maps downstream of Ptc; forskolin, the adenylate cyclase activator that functions via protein kinase A to activate destruction of Ci/Gli; and a recently identified Hh-signaling antagonist Cur61414. Lines with arrowheads represent activation and blunt-ended lines represent repression. (b-e) Luciferase-based reporter assays of Hh signaling showing inhibitory dose response on cells activated by Hh protein (10 nM) or Hh-Ag 1.2 (200 nM) of (b) 5E1; (c) forskolin; (d) cyclopamine; and (e) Cur61414. Data points represent the averages (n = 3) with standard deviations depicted by error bars.