Fluorescent Probes for Mitotic Kinase Dynamics

GFP-tagged full-length MCAK was previously reported27 (link). Point mutations within MCAK were generated using a QuikChange Site-Directed Mutagenesis kit (Stratagene). The cDNA was amplified by PCR and subcloned into p3xFLAG-myc-CMV24 (Sigma), modified pcDNA-mCherry and pFastBac1 (Invitrogen) vectors. The design of the PLK1 sensor is based on a PKC sensor40 (link): an FHA2 phospho-Thr-binding domain and a substrate peptide (LLLDSTLSINWD from Myt1) were inserted into a CFP/YFP FRET pair. The sensor was generated with CENP-B fusion to target to centromeres. The Aurora B sensor (CENP-B fusion) and PLK1 sensor (Hec1 fusion) used in experiments for Fig. 6a were described in previous reports41 (link)42 (link). All plasmid constructs were confirmed by sequencing.

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Shao H., Huang Y., Zhang L., Yuan K., Chu Y., Dou Z., Jin C., Garcia-Barrio M., Liu X, & Yao X. (2015). Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation. Scientific Reports, 5, 12204.

Publication 2015

p3xFLAG-myc-CMV24 pFastBac1

Aurora b Cdna Cenp b Centromeres Fret Hec1 Peptide Plasmid Plk1 Point mutations Site directed mutagenesis Vectors

Corresponding Organization :

Other organizations : University of Science and Technology of China, Hefei National Center for Physical Sciences at Nanoscale, Morehouse School of Medicine

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Variable analysis

independent variables

Point mutations within MCAK

dependent variables

MCAK function

control variables

GFP-tagged full-length MCAK
Positive control: PKC sensor
Positive control: Aurora B sensor
Positive control: PLK1 sensor

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