Fig. 4

MIEN1 is a novel interactor of AnxA2. a MIEN1 interacts with AnxA2 in a yeast two-hybrid screen. Positive clones were selected on high-stringency medium (synthetic dropout medium) selection markers SD/−Ade/−His/−Leu/−Trp/X-α-gal, and only true interactors can activate the expression of β-galactosidase (blue color). The left panel shows positive interaction of MIEN1 with AnxA2, but not with p53. The right panel shows the positive control p53 − T-antigen interaction, while the AnxA2 − p53 interaction served as the negative control. b Co-immunoprecipitation of AnxA2 and MIEN1 in BT-474 cells showing the interaction of the two proteins. IgG was used as isotype control antibody and the total MIEN1 and AnxA2 used for the experiment was shown by western blotting. c Confocal microscopy showing co-localization of AnxA2 and MIEN1 in cancer cells. The interaction is predominantly observed around the membrane and cytosol excluding the nucleus. HCC-70, MCF-7 images were acquired with 40x objective with 1x zoom; MDA-MB231 images were acquired with 40x objective with 2x zoom. d FRET confirms the interaction of MIEN1 and AnxA2. Lifetime decays of the donor and donor − acceptor pair was measured to be 1.75 and 1.26 ns, respectively, and the lifetime decay histograms of the donor − acceptor pair (upper) and donor (lower) are shown