Figure 2.

STIL binding stimulates Plk4 activity. (A) Cells were cotransfected with the indicated constructs and protein levels were analyzed by immunoblotting. mCherry-Mad2 serves as a transfection control. (B) Quantification of the protein levels shown in A. Bars represent the mean of three independent experiments. (C) STIL was depleted by siRNA and 24 h later doxycycline was added to induce expression of Plk4-EYFP. The immunoblot shows the relative levels of STIL and Plk4-EYFP in control or STIL siRNA–depleted cells. The graph shows quantification of the relative level of Plk4-EYFP at the centrosome of S/G2 phase cells. Bars represent the mean of at least three independent experiments, with >40 cells counted per experiment. (D) Endogenous STIL was depleted by siRNA and replaced with either Myc-GFP-STIL WT or ΔCC using the scheme outlined in Fig. 4 A. The graph shows quantification of the relative levels of Plk4 at the centrosome of S/G2 phase cells. Bars represent the mean of at least three independent experiments, with >40 cells counted per experiment. (E and F) Cells were cotransfected with the indicated constructs, and protein levels were analyzed by immunoblotting. Where indicated, lambda protein phosphatase (λ PP) was incubated with the cell lysate for 60 min before immunoblotting. (G and H) Cells were cotransfected with the indicated constructs and subjected to coimmunoprecipitation analysis with the indicated antibodies. (I) Cells were cotransfected with the indicated constructs and protein levels were analyzed by immunoblotting. All error bars represent the SEM.