Fig. 3. Eg5[Ser1033] phosphorylation in vivo. (A) U2OS cells were co-transfected with either myc-Eg5 wild type (lanes 1-3) or myc-Eg5[Ser1033Ala] (lane 4) and empty plasmid (lane 1), FLAG-Nek6 wild type (lanes 2 and 4) or FLAG-Nek6 [Lys74Met Lys75Met] (lane 3). 24 hours later, anti-Myc immunoprecipitates were subjected to immunoblot with anti-Myc (lower panel) and anti-Eg5[Ser1033-P] (top panel) antibodies. (B) U2OS cells growing exponentially were either untreated (first lane, Exp) or arrested with nocodazole (0.25 mM overnight; lane 2); arrested cells were allowed to exit mitosis in nocodazole-free medium and extracted at the times indicated (lanes 3-7). Eg5 immunoprecipitates (top two rows) and cell extracts (bottom seven rows) were analyzed by immunoblotting with the indicated antibodies. a-P-Eg5(S1033), anti-Eg5[Ser1033-P]; a-P-CDK1, anti-CDK1[Tyr15-P]; a-P-Nercc1, anti-Nercc1[Thr210-P]; a-P-Nek6, anti-Nek6[Ser206-P]. (C) Extracts from XL177 X. laevis cells were resolved by SDS-PAGE and immunoblotted with either anti-XlEg5 (left) or anti-XlEg5[Ser1046-P] (right). (D) XL177 cells growing exponentially were fixed and stained with antibodies to XlEg5 (lower two rows), XlEg5[Ser1046-P] (upper two rows) and either β- or -tubulin as indicated. DNA is stained with DAPI. Representative cells in metaphase are shown. Scale bar: 10 µm.