Figure 4: Critical sites in the STIL STAN motif for STIL/HsSAS-6 interaction and centriolar targeting of HsSAS-6.

(a) Alignment of the STAN motif within human, mouse, Xenopus and zebrafish STIL and Drosophila Ana2. Colours and symbols indicate the same as in Fig. 1c. The S/T sites tested in b are shown in green. S1061 and S1116 are highlighted in red. Grey double lines indicate the critical parts of the STAN motif identified in Supplementary Fig. 2e. (b) Alanine mutational scan for the S/T sites within the STAN motif. In vitro kinase and binding assays were performed as described in Fig. 2c except for using recombinant GST-STIL CS wild-type (WT) or non-phosphorylatable alanine mutants. (c,d) U2OS cells were treated with control siRNA (siCnt) or siRNA targeting 3′UTR of endogenous STIL (siSTIL), followed by transfection with an empty vector (–), HA-STIL WT or non-phosphorylatable mutants, 2A (mutated at S1061 and S1116 to alanine), S1061A and S1116A. The cells were fixed and immunostained with antibodies against HA and centrin (c) or HsSAS-6 (d). DNA is shown in blue. Histograms represent frequency of interphase cells with ≥4 centrioles (c) or with centriolar HsSAS-6 (d) in each condition. Insets show approximately sevenfold magnified views around the centrosome. Scale bar, 5 μm. Values are mean percentages±s.d. from three independent experiments (N>50 for each condition). *P<0.05, **P<0.01; NS, not significant (one-tailed t-test).