Fig. 4

The Smo-oxysterol interaction is conserved in vertebrates.

(A) The interaction of 20(S)-OHC beads with full-length mSmo, full-length Drosophila Smo (dSmo) or zebrafish Smo (zSmo) carrying a truncation of the intracellular C-terminal tail (zSmoΔC) was tested in the presence of free 20(S)-OHC or its enantiomer. (B) The zSmo ectodomain (which includes the CRD) can bind to 20(S)-OHC beads, but the dSmo CRD cannot. (C) Zebrafish embryos (30hpf) carrying a GFP transgene driven by the engrailed2a promoter were treated with 20(S)-OHC (50 µM) or cyclopamine (40 µM) and assessed for GFP expression by fluorescence and ptch2 expression by in situ hybridization. See Figure 4-figure supplement 1 for quantitation. (D) A binding curve (K_d ~170 nM) for the zSmo ectodomain-20(S)-OHC interaction was measured by incubating a fixed amount of protein with increasing amounts of bead-immobilized sterol. The amount of zSmo ectodomain captured on the beads (shown in the graph) was quantitated from a coomassie-stained SDS-PAGE gel shown above. (E) Binding of the zSmo ectodomain to 20(S)-OHC beads was inhibited in a dose-responsive fashion by free 20(S)-OHC but not by its enantiomer. (F and G) Coomassie-stained SDS-PAGE gels show the amount of zSmo ectodomain captured on 20(S)-OHC beads in the presence of various oxysterols (F) or Smo ligands (G).