Z-focal series of HeLa cell at metaphase stained for DNA (blue), phospho-Ser92-MAK (mitotic centromere-associated kinesin) (red), and Aurora B (green). Phosphorylation of MCAK is reduced in Bod1siRNA cells. Cells were transfected with control (this image) or Bod1 (CIL: 13378) siRNA. After 72 h, cells were treated with monastrol for 3 h and released into media containing MG132 for 1 h before fixing. Cells were stained for total mitotic centromere-associated kinesin (MCAK) population. MCAK localizes to the inner centromere in its phosphorylated form but concentrates at kinetochores in its dephosphorylated state.
Antibodies were rabbit anti-HEC1 (green) antibody (Abcam) used at 1:1,000. and anti–phospho-MCAK antibodies (red) (Andrews et al., 2004) used at 1 μg/ml, and DNA (blue). Fluorescently labeled secondary antibodies were all obtained from Jackson ImmunoResearch Laboratories. Corresponds to Fig 5D in JCB vol. 179 no. 2 187-197.
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