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. 2015 Mar 19;14(9):1447–1458. doi: 10.1080/15384101.2015.1021518

Figure 2.

Figure 2.

Tpr interacts with Aurora A during mitosis. (A) Immunoprecipitation of mitotic HeLa cell extracts incubated with anti-Tpr (mouse anti-Tpr, sc-101294 from Santa Cruz Biotechnology) or non-specific rabbit antibodies (IgG) (#2729; Cell Signaling Technology) were analyzed by immunoblotting with anti-Aurora-A (IAK1 610939 from BD Transduction Laboratories), anti-Aurora-B (ab2254, from Abcam), anti-Nup62 (m414, MMS-120R from COVANCE) and anti-Tpr (sc-101294 from Santa Cruz Biotechnology) antibodies. Numbers indicate molecular mass markers in kilodaltons. (B) Immunoprecipitates of mitotic HeLa cell extracts incubated with anti-Aurora A (IAK1 610939 from BD Transduction Laboratories) or non-specific rabbit antibodies (IgG) (#2729; Cell Signaling Technology) were analyzed by immunoblotting with the indicated antibodies (refer to Fig. 2A). (C) Co-immunostaining of Tpr and Aurora A in the cell cycle. Confocal images of HeLa cells at different mitotic stages, stained with anti-Aurora A (green) antibody and anti-Tpr (red; this antibody gives strong spindle signals in metaphase). Goat anti-mouse Alexa Fluor-488 or rabbit Rhodamine were used as secondary antibodies. Chromatin was stained with DAPI (blue). Data correspond to the sum of 3 independent experiments. (D) Aurora A is directly pulled-down by the Tpr-M domain in vitro. Tpr fragments were expressed in vitro, affinity-purified together with 6xHis-Aurora A, and separated by SDS-PAGE. Tpr fragments were prepared using the TNT Quick-Coupled Transcription/translation system (Promega) together with TranscendTM Biotin-Lysyl-tRNA (Promega). Asterisks indicate Tpr fragment (N, M and C) respectively. Streptavidin horseradish peroxidase (HRP) (1:4,000) was used for detection. Tpr fragments were untagged. Numbers indicate molecular mass markers in kilodaltons.