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. 2018 Jun 21;46(16):8216–8231. doi: 10.1093/nar/gky557

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 9. — NuSAP3 is a Kif13-1-interacting protein required for promoting chromosome segregation and for maintaining Kif13-1 stability. (A) RNAi knockdown of NuSAP3 in T. brucei, as demonstrated by western blotting of endogenously 3HA-tagged NuSAP3 in NuSAP3 RNAi cells induced for up to 4 days. TbPSA6 served as the loading control. (B) Depletion of NuSAP3 caused a severe growth defect. (C) Flow cytometry analysis of NuSAP3 RNAi cells. Insets show the percentage of cells at different cell cycle stages. (D) Effect of NuSAP3 depletion on cell cycle progression. Shown is the quantification of cells at different cell cycle stages as determined by counting the numbers of cells with different numbers of nucleus (N) and kinetoplast (K), which was stained with DAPI. A total of 200 cells were counted for each time point, and three repeats were carried out. Error bars indicate S.D. 1N*1K cells represent the cells with an abnormal-shaped nucleus and one kinetoplast. (E) Morphology of the nucleus shape of 1N1K cells in control and NuSAP3 RNAi cell line. Nuclear envelope was labeled by immunostaining of the endogenously 3HA-tagged nuclear pore protein NUP89, and DNA was stained with DAPI. N, nucleus; K, kinetoplast. Scale bar: 5 μm. (F) NuSAP3 interacts with Kif13-1 in vivo in T. brucei. Shown is the co-immunoprecipitation of PTP-tagged Kif13-1 and 3HA-tagged NuSAP3 from T. brucei cell lysate. Kif13-1-PTP alone and NuSAP3-3HA alone served as the negative controls. The co-IP was repeated three times, and similar results were obtained. (G) Effect of NuSAP3 depletion on Kif13-1 protein stability. Kif13-1 was endogenously tagged with a C-terminal triple HA epitope in NuSAP3 RNAi cell line. TbPSA6 served as the loading control. The experiment was repeated three times, and similar results were obtained. (H) Effect of Kif13-1 depletion on the level of NuSAP3 protein. NuSAP3 was endogenously tagged with a C-terminal triple HA epitope in Kif13-1 RNAi cell line. TbPSA6 served as the loading control. Similar results were obtained from three repeats.