Figure 2. Plk4 kinase activity promotes the formation of a complex between STIL and HsSAS-6.

(a) U2OS cells were treated with control siRNA or siRNA targeting 3′UTR of endogenous STIL, followed by transfection with an empty vector (–), HA-STIL full-length (FL), N (a.a. 1–1,017) or ΔSTAN (Δ1,061–1,147 a.a.). The cells were fixed and immunostained with antibodies against HA and HsSAS-6. DNA is shown in blue. Histograms represent frequency of interphase cells with centriolar HA (top) or with centriolar HsSAS-6 (bottom) 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). (b) HEK293T cells co-expressing Plk4ΔP-FLAG wild type (WT) or kinase-dead (KD) and Myc-HsSAS-6 were immunoprecipitated (IP) with HsSAS-6. The amount of expressed Plk4ΔP-FLAG WT and KD was collected by IP using FLAG beads. Total cell lysates and IPs were analysed by western blotting using STIL, HsSAS-6 or FLAG antibodies. (c–g) In vitro reconstitution of a Plk4-dependent complex formation of STIL and HsSAS-6. (c) Schematic of the method used for monitoring the interaction between Plk4-phosphorylated STIL and HsSAS-6 in vitro. Plk4ΔP-FLAG WT or KD proteins were expressed and purified from HEK293T cell using anti-FLAG beads, followed by incubation with bacterially purified STIL N3C WT or ΔSTAN mutant (in d,e), deletion mutants of STIL C (in f,g) recombinant proteins for in vitro kinase assay. After the kinase reaction, the supernatant was collected and incubated with MBP-HsSAS-6 purified from baculovirus/insect cell expression system, and thereafter pulled down using amylose resin. Input and the protein complexes pulled down with amylose resin were analysed by western blotting using STIL, FLAG, HsSAS-6 and HA antibodies. Asterisks represent cleaved products of GST-HA-STIL C or CS.