Figure 2. PLK1 interacts with and directly phosphorylates NDR1 (a).

Co-localization of NDR1 and PLK1 in mitosis. Immunofluorescence analyses of mitotic HeLa cells expressing GFP-NDR1 wild-type construct with PLK1 antibody (red) and DAPI (blue). The boxed areas are shown in the right row with magnification. The scale bar represents 10 μm.(b) Line scan of the yellow straight line in the “merge” pane of (a). Black arrowheads indicate the spindle poles.(c) Co-immunoprecipitation of Flag-NDR1 and GFP-PLK1. Lysates from the 293T cells transiently transfected to express GFP-PLK1 alone, or FLAG-NDR1 together with GFP or GFP-PLK1 were subjected to immunoprecipitation with Flag antibody or IgG. The precipitates (IP) and cell lysates were analyzed by immunoblot with antibodies against Flag and GFP. Cropped blots from one gel.(d)Pull-down assay of MBP-NDR1 using GST- or GST-PLK1-bound agarose beads as an affinity matrix.(e) In vitro kinase assay with using PLK1 kinase mixed with substrates of MBP, MBP-NDR1 kinase-death mutant (K118A) and non-phosphorylatable mutant (K118A-3A). In some case, an aliquot of reaction included PLK1 specific inhibitor BI2536. Both CBB stain gel and autoradiography are shown.