npg Fused dimerization transduces Hedgehog signal 1446 Figure 6 Phosphorylated Cos2 and Su(fu) by dimerized Fu promote the dissociation of Ci155 from the Cos2/Fu complex or Su(fu) and its nuclear translocation. (A, B-B′, C, D-D″) S2 cells were transfected with Myc-Ci155, Flag-Cos2 (A-B′) or Flag-Su(fu) (C- D′), and the indicated constructs. Cell extracts were immunoprecipitated with anti-Flag antibodies, followed by immunoblotting with indicated antibodies. Western blot for Flag-Cos2 or Flag-Su(fu) is serving as loading control, respectively. The Ci155 expression in the lysate is detected with anti-Myc antibody. (A) S2 cells were transfected with Flag-Cos2, Myc-Ci155 and Smo phosphor-mimetic mutants, which mimicked Hh gradient. (B, B′) FKBP- or Div-based Fu constructs were transfected into S2 cells with Flag-Cos2 and Myc-Ci155. (C) S2 cells were transfected with Flag-Su(fu), Myc-Ci155 and Smo phosphor-mimetic mutants. SmoSD, which mimics the high Hh concentration, but not SmoSD1 and SmoSD12, could induce the dissociation between Su(fu) and Ci155. (D, D′) The di- merized Fu (Fv2-Fu with AP20187 treatment or Div-Fu) decreased the association between Su(fu) and Ci155, compared with Fv2- FuG13V with or without AP20187 treatment, Fv2-Fu without AP20187 treatment, or Div-FuG13V. (D′′) Div-FuK* enhanced dissoci- ation of Su(fu) with Ci155, compared with Div-FuK, Div-FuKG13V and Div-FuK*G13V. (E) S2 cells were transfected with HA-Ci155, or HA-Ci155 and Flag-Su(fu), and treated with Hh or LMB, respectively, or with Hh and LMB treatment. The antibody detecting full- length Ci was used to stain for Ci155 (red), antibody recognizing Flag was used for immunostaining Flag-Su(fu) (green), and the nuclei were stained with DAPI (blue). The nuclei were circled by white line. (F, G) The percentage of S2 cells with Ci155 nuclear localization after co-transfected with Smo phosphor-mimetic mutants to mimic Hh gradient (F), or with dimerized Fu. FKBP- or Div- based dimerized Fu could antagonize Su(fu) and promote Ci155 nuclear translocation (G). In F and G, pUAST and Hh treatment served as negative and positive control, respectively. Cell Research | Vol 21 No 10 | October 2011