Fig. 7. Phosphorylation of STIL by Plk4 triggers centriole duplication.
(A) Flag-STIL full-length or 5A mutant (S871A/S873A/S874A/S1116A/T1250A) expressed in HEK293T cells and immunoprecipitated with anti-Flag antibodies was incubated with bacterially expressed Zz-Plk4 in the presence of [γ-32P]-ATP. In vitro kinase assay with Flag-STIL or Plk4 alone served as a control. Kinase assays were analyzed by SDS-PAGE, Coomassie Blue staining and autoradiography. (B) Co immunoprecipitation of Flag-STIL wt/5A and Myc-Plk4. Lysates from HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation using anti-Flag antibodies. Input and IP samples were analyzed by western blotting with antibodies against Flag- and Myc-tag. The asterisk marks an unspecific band recognized by the anti-Myc antibody. The dividing lane indicates grouping of images from different parts of the same gel, as an intervening lane was removed for presentation purposes. (C) U2OS cells transiently expressing Flag EV, Flag-STIL wt or Flag-STIL 5A were analyzed by indirect immunofluorescence using staining with anti-CP110 and mouse anti-Flag antibodies 72 h after transfection. The number of transfected cells with more than four centrioles was determined based on CP110 staining. Values in the graph are mean percentages±s.d. from three independent experiments, 50 transfected cells were analyzed in each experiment (***P<0.001, two-tailed t-test). Representative images are shown for control, Flag-STIL wt and 5A-transfected cells. Scale bars: 10 µm (merge), 2 µm (magnifications). Western blotting using antibodies against Flag and α-tubulin was performed to visualize expression of STIL constructs as indicated.