Hh promotes CL-II phosphorylation through Gish.

(A) Diagram of Smo with PKA, CK1, and Gprk2 sites indicated by different colors. Black and gray boxes denote the seven transmembrane domains and the Smo auto-inhibited domain (SAID), respectively. (B) In vitro kinase assay using recombinant CK1 and GST-Smo fusion proteins carrying wild-type (GST-Smo_601-700) or mutated CL-II site (GST-Smo_601-700CL-IISA). (C, D) Western blots of lysates from S2 cells transfected with the indicated constructs, treated with or without the indicated dsRNA and Hh-conditioned medium. (E, F, L) Western blots of immunoprecipitation experiments from lysates of S2 cells transfected with the indicated constructs. Cells were grown in the presence or absence of Hh stimulation for 24 h and exposed to MG132 (50 μM) for 4 h before harvest. (G, H) S2 cells transiently (G) or stably (H) expressing Myc-Smo were treated with control RNAi, Gish RNAi, or D4476. Cells were grown in the presence or absence of Hh stimulation for 24 h and exposed to MG132 (50 μM) for 4 h, followed by immunoprecipitation and western blot analysis.