Figure 2

EG5 is a novel PP2A binding protein. (A) Silver staining of FLAG pull down assay. HeLa cells stably expressing FLAG-PP2A/Cα and control cells were lysed and incubated with FLAG antibody bound to beads, and FLAG peptide elutes were subjected to electrophoresis. (B,C) PP2A and EG5 showed association in HeLa and BT549 cells. Cell lysate was subjected to immunoprecipitation with anti-PP2A/A or anti-EG5 antibody, then evaluated with anti-EG5 or anti-PP2A/A antibody. (D) PP2A and EG5 interact in vitro. Purified His-PP2A/Cα was incubated with FLAG-EG5 from SF9 cells or with other control proteins, and the immunoprecipated complexes were identified with anti-FLAG antibodies. (E) Constructs of FLAG-tagged EG5 fragments (Head 1 ~ 350aa, Stalk 351 ~ 550aa, Tail-1551aa ~ 750aa, and Tail-2 751 ~ 1057aa). (F) The C terminal (Tail-2) of EG5 interacts with PP2A/C.EG5 and fragments expressed in HEK293T cells were incubated with His-PP2A/C, and precipitated with Ni-Beads. Association was evaluated with anti-FLAG antibody and Western blot. (G) EG5 associates with the PP2A/B55α complex. HeLa cell lysate was subjected to immunoprecipitation with anti-PP2A/B55α antibody, and evaluated with anti-EG5 and anti-PP2A/C antibodies. For the original uncropped western blots and gel data, see Supplementary information.