a, Primary structure of Ptch1. Residues 619–720 and 1189–1447 were removed in Ptch1*. b, Hh signaling in Ptch1−/− MEFs transfected with Ptch1 or Ptch1* and response to wild-type or mutant Shh-N ligand via luciferase activity. Shh-N in conditioned medium and transiently expressed Ptch1 were detected by western blotting. c, Pull-down assay of his-tagged Shh-N or native Shh-N with Ptch1* at different molar ratios detected by Coomassie-staining. The assay was reproduced three times with the similar results. d, Palmitoylated Shh-N stimulates Hh signaling but unmodified Shh-N does not. Shh-Light II cells were treated with various concentrations of Shh-N varients, and Hh signaling was measured using luciferase activity. Data (panels b and d) are mean ± s.d. (n=3).