Fig. S6.

Shp2 localizes to the kinetochore through binding to Plk1. (A) GFP-Plk1 and various Flag-tagged Shp2 mutant plasmids were cotransfected into 293T cells. Forty-eight hours after transfection, cells were treated with nocodazole (200 ng/mL) for 16 h. Cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting with anti-GFP antibody. Blots were stripped and reprobed with anti-Flag antibody to determine the expression of various Shp2 constructs. Equal GFP-Plk1 expression levels were verified by immunoblotting with anti-GFP antibody using the whole-cell lysates. (B) GST and various GST–Shp2 fusion proteins were purified by Glutathione-Sephorose 4B beads. Immobilized proteins (5 μg) on beads were incubated with whole-cell lysates (500 μg) prepared from mitotic HeLa cells. The resulting complexes were washed, resolved by SDS/PAGE, and subjected to immunoblotting with anti-Plk1 antibody. (C) Various Flag-tagged Shp2 mutant plasmids were transfected into HeLa cells. Cells were immunostained with anti-Flag antibody and CREST autoimmune serum. Nuclei were counterstained with DAPI. Kinetochores in prometaphase/metaphase cells were examined under a confocal microscope.