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. 2013 May 21;4:1882. doi: 10.1038/ncomms2879

Figure 4. Plk1–Ser99 phosphorylation is indispensable for Plk1 activation pathway via 14-3-3γ.

Figure 4

(a) Myc–Plk1 WT and S99A were introduced in Tet-On HeLa cells and then stained with anti-Myc (green), anti-γ-tubulin (magenta) and CREST (red). (b) The effect of 14-3-3γ silencing on subcellular localization of endogenous Plk1. Arrows indicate the position of centrosomes. (c, d) The evaluation of mitotic protein phosphorylation after the replacement of endogenous Plk1 with exogenous Myc–Plk1, using the indicated Tet-On HeLa cell line. Owing to silent mutation on the gene coding each Myc–Plk1, the induced messenger RNA is resistant to Plk1 siRNA target sequence #1 (siPlk1, #1). Tet-On HeLa cells expressing GFP were used as a negative control (d). (e,h) The evaluation of mitotic index after the replacement. In h, Tet-On cells incubated without doxycycline after the treatment with siPlk1 (#1) are used as negative controls. (f) The effects of the replacement on KT-associated BubR1. Scale bars, 10 μm. (g) Each Myc–Plk1 construct was immunoprecipitated from mitotic Tet-On cells and then subjected to immunoblotting or IP kinase assays.