Fig. 3: Enhancing BubR1-Plk1 or BubR1-B56 binding could bypass the requirement of the double phosphorylation.
From: Self-priming of Plk1 binding to BubR1 ensures accurate mitotic progression
a KD measured by ITC of recombinant Plk1 with phospho-modified peptides. b Representative images of mitotic cells transfected with siRNA oligos against BubR1 and RNAi-resistant constructs expressing YFP-BubR1. The cells were released from RO3306 into the medium with nocodazole (200 ng/ml) for 45 min before fixation and staining with corresponding antibodies. Scale bar is 10 µm. c Quantification of kinetochore signals of Plk1 against YFP-BubR1 from (b). Mean values of at least 150 kintochores from at least 10 cells for each condition were presented. The red line indicates the mean value which was set to 1 for BubR1 ΔKARD sample and the rest was normalized to it. Bar is standard error of the mean. Mann–Whitney U-test was applied. ns means not significant; ****P < 0.0001. d Representative stills of unperturbed mitosis recorded by live cell imaging. The cells were transfected with siRNA oligos against luciferase as control or siRNA oligos against BubR1 with RNAi-resistant constructs expressing YFP-BubR1. mCherry-H3 expressing plasmid was co-transfected for chromosome indication. Scale bar is 10 µm. e Quantification of the mitotic time from NEBD to anaphase from (d). Each circle represents the time from NEBD to anaphase of a single cell. Red line indicates the median time. The number of cells analyzed per condition is indicated above (n = X). Mann–Whitney U-test was applied. *P < 0.1; ***P < 0.001; ****P < 0.0001.
