CL-II phosphorylation depends on PKA and cell surface localization of both Smo and Gish.

(A) Diagrams of wild-type Gish and its mutant derivatives. (B) Immunostaining of S2 cells transfected with the indicated Gish constructs. (C) Western blots of immunoprecipitation experiments from lysates of S2 cells treated with dsRNA targeting the gish 5’ UTR and transfected with Myc-Smo together with the indicated Gish 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. (E, F) S2 cells were transfected with Myc-Smo or Myc-Smo-Ub and stimulated with or without Hh, followed by immunostaining to visualize cell surface Smo (top panels in E) or total Smo (bottom panels in E), or by immunoprecipitation and western blot analysis with the indicated antibodies. (G) S2 cells stably expressing Myc-Smo were treated with or without Hh-conditioned medium and H89 and exposed to MG132 (50 μM) for 4 h, followed by immunoprecipitation and western blot analysis by the indicated antibodies. (H) Western blots of immunoprecipitation experiments from lysates of S2 cells transfected in indicated Smo 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. (I, J) S2 cells stably expressing Myc-Smo were treated with different levels of Hh or the same level of Hh for different periods of time, followed by immunoprecipitation and western blot analysis by the indicated antibodies. Quantification of Smo4P and P687 signals is shown to the right. The signal intensities at 100% Hh or 24 h are defined as 100. Data are means ± SD from three independent experiments.