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. Author manuscript; available in PMC: 2015 Aug 13.
Published in final edited form as: Discoveries (Craiova). 2014 Jun 30;2(2):e16. doi: 10.15190/d.2014.8

Figure 2. Screening validation: PLK1 kinase forms a complex with FOXO3 and other FOXO family members (FOXO1 & FOXO4).

Figure 2

A. HeLa cells were transfected with either pcDNA3-EV-FLAG-HA (Control) or pcDNA3-FOXO3-FLAG-HA and exogenous FOXO3 was immunoprecipitated by using an anti-FLAG antibody. The results show that exogenous FOXO3 is co-immunoprecipitated with endogenous PLK1 in HeLa cells. B. HeLa cells were transfected with either pcDNA3-EV-FLAG-HA (Control) or pcDNA3-FOXO3-FLAG-HA and endogenous PLK1 was immunoprecipitated by using an anti-PLK1 antibody. The results show that endogenous PLK1 is co-immunoprecipitated with exogenous FOXO3 in HeLa cells (revers immunoprecipitation). C. HeLa cells were transfected with either pcDNA3-EV-FLAG (Control), pcDNA3-FOXO1-FLAG, pcDNA3-FOXO3-FLAG and pcDNA3-FOXO4-FLAG. Exogenous FOXO1/FOXO3/FOXO4 was immunoprecipitated by using an anti-FLAG antibody. The results demonstrate that all three FOXO family members can form a complex with endogenous PLK1; D. Purified GST-FOXO3 protein was incubated with lysates from PLK1-FLAG transfected HeLa cells. GST pull-down reveals that GST-FOXO3 forms a complex with PLK1 (detected with an anti-FLAG antibody). GST is used as a negative control, while 14-3-3, a known scaffold protein of FOXO3, is used as a positive control. Results obtained with several different concentrations of NP40 detergent are shown. E. Asynchronous and synchronized proliferating HeLa cells in different phases of the cell cycle were used to evaluate the endogenous-endogenous interaction between FOXO3 and PLK1. Cells were synchronized with Nocodazole and released. Cells collected at 0, 5, 10, 15 and 20h after Nocodazole release were evaluated for cell cycle phases by analyzing the Cyclins expression (B1, A), which are particularly expressed in specific phases of the cell cycle (see right panel of the lower figure). PLK1 is also expressed at highest levels in mitosis and can itself be used as an indicator of cell cycle phase. Immunoprecipitated endogenous FOXO3 co-immunoprecipitates with endogenous PLK1. While the FOXO3-PLK1 complex can be seen during all phases of the cells cycle and in asynchronous cells, most PLK1 is bound to FOXO3 during the M/G2-M phases of the cell cycle. This is probably due to the fact that PLK1 has the highest expression during these phases of the cell cycle; PLK1 activity is determined by measuring the phosphorylation of NPM at Ser4 (site phosphorylated by PLK1)42.