Fig. 6.

Important role of phosphorylation of Y¹⁶⁶ and Y⁴⁴⁵ for the activation of Plk1. Shp2 E76K cDNA and the control vector were transfected into HeLa cells. Shp2 E76K-overexpressing cells were then transfected with Plk1 shRNA targeting 3′UTR. These Plk1 knockdown cells were subsequently transfected with WT GFP-Plk1, GFP-Plk1 mutants with Y166F or Y445F mutations, or the control vector. (A) Forty-eight hours later, cells were treated with nocodazole (200 ng/mL) for 16 h, and the percentages of mitotic cells were determined by propidium iodide (PI) and p-histone H3 double staining followed by FACS analyses. (B) Metaphase cells with chromosomal misalignment were counted. More than 50 mitotic cells for each group were examined. (C) Whole-cell lysates prepared from mitotic cells were examined by immunoblotting as indicated.