Figure 3: Plk1 phosphorylation of FoxM1b regulates its interaction with Rb.

(A) Rb interacts with an underphosphorylated form of FoxM1. Cell extracts prepared from MCF7 cells were immunoprecipitated with IgG, Rb, and FoxM1 antibody. Immunoprecipitates were subjected to western blot analysis with pan FoxM1 antibody. (B) A phospho-specific antibody (P3) was generated using a phospho-peptide corresponding to the Plk1-sites in FoxM1 and this antibody detects FoxM1 in CBP immunoprecipitate. Extracts of MCF7 cells were immunopreciptated with IgG, Rb or CBP antibody and subjected to western blotting that was probed with the P3 antibody or pan-FoxM1 antibody. (C) Extracts of MCF7 cells were immunoprecipitated with either IgG or the P3-ab. The immunoprecipitates were analyzed by western blotting with CBP-ab or Rb-ab. (D) MCF7 cells were treated with a Plk1 inhibitor (BI 2536) for 24 h at 100 nM concentration or with DMSO. Cell extracts were immunoprecipitated with either IgG or FoxM1-antibody and western blotted for FoxM1, Rb and DNMT3b. (E) Exogenous Plk1 kinase expression inhibits Rb binding to FoxM1. Wild type FoxM1 was expressed in MCF7 cells together with either kinase active PLK (PLK) or kinase dead PLK (KDPLK). Extracts were immunopreciptated with GFP antibody and western blotted for Rb.