Figure 4
Identification of Ser288 of CAP-H2 as a Plk1 phosphorylation site. (A) 293 cells were co-transfected with Flag-CAP-H2 and GFP-Plk1-T210D. Cell lysates were incubated in the absence or presence of lambda protein phosphatase. The lysates were subjected to phos-tag SDS-PAGE and immunoblot analysis. (B) 293 cells were co-transfected with indicated plasmids. Cell lysates were subjected to phos-tag SDS-PAGE and immunoblot analysis using indicated antibodies. Hyper phosphorylated form of Flag-CAP-H2 (red arrow) was diminished in S288A mutant. (C) Recombinant GST-CAP-H2 was incubated with ATP in the absence or presence of His-Plk1 and BI2536. The reaction products were analyzed by immunoblotting with the indicated antibodies. (D) HeLa cells were arrested at mitosis by nocodazole and then were treated with BI2536 for 2 h. The lysates were subjected to immunoprecipitation with anti-CAP-H2. The lysates and immuneprecipitates were immunoblotted with the indicated antibodies. A ratio of p-CAP-H2-Ser288/CAP-H2 was determined by the densitometric analysis using Fusion-CAPT-Software. The ratio of p-CAP-H2-Ser288/CAP-H2 in asynchronous cells was defined as 1.0.
