Plk1 phosphorylation negatively regulates central spindle recruitment of Cep55. (A) HeLa cells treated with DMSO, 1 µM BI2536, 5 µM flavopiridol, or 1 µM ZM447439 for 25 min were stained for DNA with DAPI and mouse anti–α-tubulin, rabbit anti-Cep55 (green), and goat anti-Plk1 (red). (B) HeLa cells transiently expressing EGFP-PRC1 or the Plk1-binding defective ST602AA mutant were transfected with control or PRC1 3′ untranslated region (UTR) siRNAs for 36 h. Cells were stained for DNA with DAPI, sheep anti-Cep55 (false-colored green in the merge), and goat anti-Plk1 (red). PRC1 was visualized using EGFP fluorescence. (C and D) Cep55 (C) and MKlp1 (D) complexes were isolated using sheep antibodies from mitotic HeLa cells treated with DMSO (control), 1 µM BI2536, or 1 µM ZM447439 for 25 min and then Western blotted. The asterisks indicate cross-reactivity to the heavy chain of the antibody used for immunoprecipitation. Western blot markers are given in kilodaltons. Bars, 10 µm.