Figure 6.

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.