Figure 2. 14-3-3γ binds to Plk1 and stimulates its catalytic activity.

(a,b) Identification of proteins bound to GST-14-3-3γ specifically in mitotic extract. Mitosis- and 14-3-3γ-specific binding proteins (bands 1-4) are shown to the right (a). The presence of Plk1 was also checked by immunoblotting with anti-Plk1 (b). An asterisk or arrow indicates the position of GST-14-3-3γ or Plk1, respectively (b). (c) IP with anti-Plk1 from interphase (I) or mitotic (M) HeLa cell extract. The same amount of mouse IgG was used as a negative control. (d) GST pull-down assays using each subtype of GST-14-3-3 proteins form mitotic cell extract. (e–i) The effect of 14-3-3γ silencing on catalytic activities of mitotic kinases. Anti-Plk1 (e, i), anti-AurA (f), anti-AurB (g) or anti-Cyclin B1 immunoprecipitates (h) were subjected to IP kinase assays. Each immunoprecipitates and cell lysates (input) were also subjected to immunoblotting. In f, 8–128 ng μl⁻¹ of purified 14-3-3γ was added in mitotic extract before IP. (j) The effects of 14-3-3γ or Plk1 depletion on intracellular phosphorylations.