Supplementary Figure 5: Riquiqui, Minibrain and Warts physically interact with each other and Minibrain phosphorylates Warts.

(a) S2 cells were transfected with the indicated plasmids and immunoprecipitations performed using anti-HA antibodies. Subsequently, immunoprecipitates and input lysates were subjected to SDS-PAGE and Western blotted to reveal the indicated proteins. (b–d) Western blot analysis of lysates from S2 cells transfected with the indicated plasmids and Western blotted to reveal the indicated proteins. In (b) Riq or Mnb were expressed in S2 cells in the presence or absence of dsRNA specific for Riq or Mnb, respectively. In (c) Mnb did not induce changes in Hpo or Sav mobility. In (d) Mnb, but not Hipk influenced Wts mobility. (e) Kinase assays performed using recombinant GST-Wts1 or GST-CACYBP as substrates and either Mnb or Mnb K386R immunoprecipitated from S2 cells. Immunopurified and recombinant proteins were incubated alone or together in kinase buffer containing γ³²P-ATP and subjected to SDS-PAGE (upper panel). Western blotting was used to detect input proteins (lower panels). Mnb phosphorylated GST-Wts1 but not the negative control substrate GST-CACYBP. (f) V5-tagged Wts was expressed in the presence or absence of dsRNA specific for Riq or Mnb in S2 cells. Cells were lysed and lysates were immunoblotted using antibodies to V5 and to Tubulin as a loading control. Wts levels were unchanged when Riq or Mnb were depleted from cells by RNAi.