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. 2017 Jul 14;49(7):e354. doi: 10.1038/emm.2017.92

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Copyright © 2017 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

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The localization and purification of BirA–CKAP2 and its phosphorylation sites. (a) The expression of BirA–EGFP or BirA–CKAP2 was confirmed by western blot analysis with anti-Myc antibody. (b) The ectopic expression of BirA–EGFP or BirA–CKAP2 in NIH 3T3 cells that were co-immunostained with streptavidin (red) and myc (green) and merged with DAPI (blue) after treatment with biotin. Representative images show that biotinylated CKAP2 localized to spindles, centrosomes and chromosome. Scale bar, 5 μm. (c) Biotinylated proteins purified using affinity purification with streptavidin and visualized by Coomassie staining. (d) The human and mouse homologs of CKAP2 were compared in terms of their amino-acid sequences with the identified phosphorylated sites in humans and the corresponding sites in mice (red). (e, f) The peptides including T603 and S608 were phosphorylated by the CDK1/cyclin B1 complex in vitro and were analyzed using ETD mass spectrometry. ETD, electron transfer dissociation.