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Fig. 2. PP4 regulates Smo phosphorylation. (A) PP4RNAi elevates Smo phosphorylation. S2 cells were co-transfected with UAS-Myc-Smo and UAS-GFP, and treated with Hh-conditioned or control medium, or treated with OA, PP4 dsRNA, PP4R dsRNA, Mts dsRNA, Wdb dsRNA or GFP dsRNA. Cell extracts were immunoprecipitated and blotted with anti-Myc to detect Smo phosphorylation. Arrow indicates hyperphosphorylated forms of Smo and arrowhead indicates hypophosphorylated and unphosphorylated forms. The efficiency of knockdown of individual phosphatase is shown in Fig. S2A in the supplementary material. (B) PP4 downregulates the Hh-induced Smo phosphorylation. S2 cells were transfected with indicated constructs and treated with or without Hh. The levels of Smo phosphorylation were examined by immunoprecipitation. The expression levels of HA-PP4 or HA-Mts were shown by probing cell lysates with HA antibody. (C) PP4 interacts with Smo C-tail. Extracts from S2 cells expressing indicated constructs were immunoprecipitated with Myc antibody followed by western blot with HA antibody. The expressed proteins were shown by probing immunoprecipitates with anti-Myc, or probing the cell lysates with anti-HA. The asterisk indicates the slow mobility of the Myc-SmoCT (Jia et al., 2003). (D) SmoCT truncations interacting with PP4. See immunoprecipitation results in Fig. S3A in the supplementary material. (E) Deletion of the PP4-binding domain in Smo elevates its phosphorylation. Extracts from S2 cells expressing Myc-Smo or Myc-Smo626-678 and treated with or without Hh were immunoprecipitated and blotted with the anti-Myc antibody. (F-G') Wing discs expressing UAS-Myc-Smo or UAS-Myc-Smo626-678 by ap-Gal4 were immunostained to show Myc expression in F' and G', and ectopic dpp-lacZ expression in F and G. Expressing Myc-Smo626-678 induced higher level of ectopic dpp-lacZ expression (arrowhead in G), compared with expressing Myc-Smo (arrowhead in F). (H) Hh downregulates Smo-PP4 interaction. Extracts from S2 cells expressing Flag-PP4 with or without Hh treatment were incubated with the bacterially expressed GST or GST-Smo557-686 fusion proteins. The bound PP4 proteins were analyzed by western blot with Flag antibody. The middle panel shows the GST and GST-Smo fusion proteins. The lower panel indicates the equal amount of input PP4. (I) Smo-PP4 interaction is attenuated by Cos2 RNAi. S2 cells were transfected with HA-PP4 and Flag-SmoCT and treated with or without Cos2 dsRNA. The SmoCT-bound PP4 was examined by immunoprecipitation with anti-Flag and western blot with anti-HA. Equally expressed PP4 proteins were ensured by probing the lysates with anti-HA. The efficiency of Cos2 RNAi is shown in Fig. S2B in the supplementary material. (J) PP4 directly interacts with Cos2MB and Cos2CT. Bacterially expressed and purified GST, GST-PP4, His-Cos2MB and His-Cos2CT were used for pull-down assay. Antibodies used for western blots are indicated.

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