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

Figure 6. Akt participates in Plk1 activation process via 14-3-3γ by regulating Plk1–Ser99 phosphorylation.

Figure 6

(a, b) The effect of Akt silencing on Plk1 phosphorylation, Plk1 interaction with 14-3-3γ (a) and Plk1 catalytic activity (b). (c, f) Using Tet-On HeLa cells expressing Flag-Akt1 WT (c) or Myc–Plk1 T210D (f), we performed the combination of DTB synchronization and the treatment with Akt1- and Akt2-specific siRNAs (siAkt1/2) as described in Hirota et al.16 Just after release from second thymidine block, the cells were cultured with (+) or without (−) doxycycline in the medium. Each protein level was checked by the immunoblotting with anti-Akt and anti-Plk1 (c) or anti-Myc (f). (d) Live-cell imaging analyses using H2B-GFP-expressing HeLa cells (n≥130 from two independent experiments). (e) The effect of Akt silencing on bipolar spindle formation. (g) KD or CA Flag-Akt1 was introduced in Tet-ON HeLa cells, and then mitotic phosphorylation of Plk1 was evaluated. (h,i) In vitro Akt kinase assays using depleted/reconstituted HeLa cell extracts (h) or anti-Plk1 immunoprecipitates (i) were performed as described in ‘Methods’.