Fig. 6. PP2A downregulates Ci phosphorylation and blocks Ci proteolytic processing. (A) Ci^FL phosphorylation is upregulated by PP2A RNAi. S2 cells were transfected with Flag-Ci and treated with OA or indicated dsRNA. Cell extracts were subjected to direct western blot with anti-Flag antibody. Arrow indicates hyperphosphorylated forms of Ci and arrowhead indicates the hypophosphorylated or unphosphorylated forms. β-Tubulin serves as loading control. The knockdown efficiency of individual phosphatase was estimated by the method used for Fig. 2A. (B) PP2A downregulates Ci^FL phosphorylation. S2 cells were transfected with Flag-Ci alone or along with indicated HA-tagged phosphatase and treated with or without OA. Cell lysates were probed with anti-Flag or anti-HA antibodies. (C) A disc shows the wild-type Ci^FL staining. (D) A wing disc shows the Ci^FL stabilization by the treatment of proteasome inhibitor MG132. (E,F) Wing discs expressing UAS-WdbRNAi by ap-Gal4 were treated with or without MG132 and stained to show Ci^FL. Arrowhead in E indicates the destabilized Ci^FL by Wdb RNAi. Arrowhead in F indicates that the destabilized Ci^FL by Wdb RNAi was restored by MG132 treatment. (G-H') Wing discs bearing smo³ clones and expressing UAS-HA-Ci^FL alone or along with UAS-Wdb by MS1096 Gal4 were stained to show the expression of GFP (green) and hh-lacZ (red). Arrowheads in G and H indicate smo³ clones that are marked by the lack of GFP expression. Arrowheads in G' and H' indicate the hh-lacZ expression in smo³ cells. (I) Western blot analysis of protein extracts from wing discs expressing UAS-HA-Ci^FL or co-expressing UAS-HA-Ci^FL with UAS-Wdb using the MS1096 Gal4. Protein extracts were prepared from 400 wing discs, immunoprecipitated and blotted with HA antibody. (J) A model for the involvement of PP4 and PP2A in Hh signaling. PP4 negatively regulates Hh signal transduction by antagonizing the phosphorylation of Smo. PP2A positively regulates Hh pathway by counteracting kinases to downregulate Ci^FL phosphorylation and attenuate its proteasome-mediated processing.