Assessing whether Smoothened is the molecular target of the Hh agonist. (a) The number of counts per minute (cpm) precipitated from an immunocomplex binding assay of 293T cells incubated with [3H]-Hh-Ag 1.5. Anti-HA (columns 1,3-9) or anti-v5 (column 2) immunocomplexes were isolated from 293T cells that were untransfected (column 1) or transfected with expression constructs encoding a rat β2-adrenergic receptor cDNA carrying a v5 epitope tag (column 2; βAR), or an HA-epitope-tagged Smo cDNA (columns 3-9). Prior to cell lysis and immunoprecipitations, these cells were incubated with 5 nM [3H]-Hh-Ag 1.5 alone (columns 1-3) or with 5 nM [3H]-Hh-Ag 1.5 in the presence of 5 μM of various unlabeled compounds (columns 4-9): Hh-Ag 1.5 (column 4); an inactive Hh-Ag 1.1-derivative containing a two-carbon linker instead of the cyclohexane ring (Ag control, column 5); the potent natural product Hh-signaling-inhibitor derivative KAAD-cyclopamine (column 6); the inactive natural product tomatadine (Antag control 1, column 7); the synthetic Hh-signaling inhibitor Cur61414 (column 8); or an inactive derivative of Cur61414 (Antag control 2, column 9). Standard deviations (n = 2) are represented by error bars. (b,c) Filtration membrane-binding assay using [3H]-Hh-Ag 1.5 (2 nM) and membranes (approximately 5 μg protein) from 293T cells transfected with different cDNA constructs. (b) Bound [3H]-Hh-agonist (cpm) when using membranes from cells transfected with murine Smo (column 1); GFP (column 2); rat β2-adrenergic receptor (βAR, column 3), and murine Ptc1 (column 4). A no-membrane control (column 5) is also included, to demonstrate the level of nonspecific binding associated with the filtration plate apparatus. (c) A competition experiment using membranes from cells transfected with murine Smo and incubated with [3H]-Hh-Ag 1.5 (2 nM) in the presence of various unlabeled compounds: no competitor (-, column 1); 2 μM unlabeled Hh-Ag 1.5 (column 2); 2 μM inactive Hh-Ag 1.1 derivative (Ag control, column 3); KAAD-cyclopamine (column 4); tomatadine (Antag control 1, column 5); Cur61414 (column 6); or an inactive derivative of Cur61414 (Antag control 2, column 7). Standard deviations (n = 4) are represented by error bars.