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. 2015 Sep 25;4:e09384. doi: 10.7554/eLife.09384

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© 2015, Hehnly et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 6. — (A) Direct binding of purified Gravin GST-fusion proteins (first and last amino acid number of each fragment is denoted above each lane) with recombinant V5-tagged Aurora A kinase (generated by in vitro transcription and translation). (Top) Immunoblot detection of Aurora A in complex with GST-Gravin fragments. (Bottom) Ponceau stained blot shows protein expression levels. (B) Spinning disc confocal image (maximum projection) of a metaphase cell (control vector, left) stained for (green) Aurora A and (blue) DNA. The subcellular rearrangement of Aurora A following over-expression of the Aurora A disruptor fragment (right). DNA is shown in blue (DAPI). (C) Live cell imaging of HeLa cells stably expressing H2B-GFP were monitored through mitosis from the time of DNA condensation until anaphase exit. Shown are (top panels) a representative control cell and (bottom panels) a cell expressing the Aurora A disruptor fragment. Bar, 5 μm. (D) Amalgamated data from multiple cells stably expressing H2B-GFP and monitored for time spent in mitosis. Control cells (n = 95 cells) and Aurora A disruptor expressing cells (n = 55 cells) were from three independent experiments (**p < 0.001 ). (E–I) Live cell imaging time courses (0–40 min) of cells stably expressing H2B-GFP transfected with (top) control shRNA and (second) Gravin shRNA. Rescue experiments as indicated with (third) murine Gravin; (fourth) murine Gravin T766A; and (fifth) murine GravinΔPKA. Bar, 5 μm. (F) Amalgamated data from multiple cells treated with control or Gravin shRNA, and rescued with murine Gravin as shown in E. These cells were stably expressing H2B-GFP and monitored for time spent in mitosis. Total cell numbers are indicated on graph (from three independent experiments, **p-values <0.001). (G–I) Models depicting the kinase-binding properties of the Gravin mutants used in time course experiments E and F: rescue with intact Gravin (G), Gravin T766A (H), and GravinΔPKA (I).

DOI: http://dx.doi.org/10.7554/eLife.09384.011

Figure 6—figure supplement 1. — (A) Direct binding of purified Gravin GST-fusion proteins (first and last amino acid number of each fragment is denoted above each lane) with recombinant V5-tagged Aurora A kinase (generated by in vitro transcription and translation). (Top) Immunoblot detection of Aurora A in complex with GST-Gravin fragments. (Bottom) Ponceau stained blot shows protein expression levels. (B) Spinning disc confocal image (maximum projection) of a metaphase cell (control vector, left) stained for (green) Aurora A and (blue) DNA. The subcellular rearrangement of Aurora A following over-expression of the Aurora A disruptor fragment (right). DNA is shown in blue (DAPI). (C) Live cell imaging of HeLa cells stably expressing H2B-GFP were monitored through mitosis from the time of DNA condensation until anaphase exit. Shown are (top panels) a representative control cell and (bottom panels) a cell expressing the Aurora A disruptor fragment. Bar, 5 μm. (D) Amalgamated data from multiple cells stably expressing H2B-GFP and monitored for time spent in mitosis. Control cells (n = 95 cells) and Aurora A disruptor expressing cells (n = 55 cells) were from three independent experiments (**p < 0.001 ). (E–I) Live cell imaging time courses (0–40 min) of cells stably expressing H2B-GFP transfected with (top) control shRNA and (second) Gravin shRNA. Rescue experiments as indicated with (third) murine Gravin; (fourth) murine Gravin T766A; and (fifth) murine GravinΔPKA. Bar, 5 μm. (F) Amalgamated data from multiple cells treated with control or Gravin shRNA, and rescued with murine Gravin as shown in E. These cells were stably expressing H2B-GFP and monitored for time spent in mitosis. Total cell numbers are indicated on graph (from three independent experiments, **p-values <0.001). (G–I) Models depicting the kinase-binding properties of the Gravin mutants used in time course experiments E and F: rescue with intact Gravin (G), Gravin T766A (H), and GravinΔPKA (I).

DOI: http://dx.doi.org/10.7554/eLife.09384.011