Fig. 1. One of the Hh-dependent Cos2 phosphorylation sites is located within the Fu-binding domain of Cos2. (A) Western blot analysis showing the differences in the electrophoretic changes of the Fu and Cos2 proteins in S2 cells treated with Hh-N-conditioned medium or okadaic acid (OA). (B) In fu-targeted RNAi cells, the Hh-dependent electrophoretic shift of Cos2 is impaired, whereas it is not impaired upon pka or sgg RNAi treatment. Cellular extracts were subjected to western analysis using Cos2, Fu, Sgg and PKA antibodies. (C) ptc RNAi induced a Cos2 electrophoretic shift, whereas double RNAi treatment targeting ptc and fu blocked this shift. (D) Structure of wild-type Cos2 protein and Myc-tagged deleted constructs. All constructs contain a Myc tag at the C-terminal end that preserved Cos2 function when expressed in transgenic animals. (E) Cells were transfected with fu-V5 and with different cos2-myc constructs (described in D). Immunoprecipitates were analysed for the presence of Fu with an anti-V5 antibody (upper panel) or for the presence of Cos2 with an anti-Myc antibody (lower panel). The large arrow indicates the smallest cos2-myc (543-605) construct capable of interacting with the Fu protein. (F) S2 cells expressing cos2-WT-myc or cos2-572A-myc were treated with OA. Arrows indicate the different phosphorylated states of the proteins. WB, western blot.