Mapping the CPC-binding domain using an MKLP2 rigor mutant trap assay. (A) HeLa cells transfected with full-length (amino acids 1–890) GFP-MKLP2 E413A ATPase defective rigor mutant and C-terminal truncations to positions 850, 750, and 650 were fixed and then stained for Aurora B (red) and DNA (blue). GFP fluorescence for MKLP2 (green) was visualized directly. (B) Comparison of amino acid truncations from MKLP2 position 640 to 690. Cells were stained for DNA, Aurora B, and PRC1 to mark the central spindle. GFP fluorescence for MKLP2 was visualized directly. Representative cells in anaphase are shown. (C) HeLa T-REx GFP-MKLP2^535-718 cells were left uninduced or induced with doxycycline for 18 h and then fixed and stained for INCENP and Aurora B. Representative cells in metaphase, anaphase A, and anaphase B are shown. (D) HeLa T-REx GFP-MKLP2^535-718 cells were left uninduced or induced with doxycycline for 18 h and then arrested in mitosis with nocodazole for 18 h. Cells were forced into anaphase using 5 µM flavopiridol to rapidly inhibit CDK1, and samples were collected at the times indicated. MKLP2 complexes were isolated by GFP Iimmunoprecipitation and blotted for the proteins shown in the figure.