Figure 2.
NuSAP is phosphorylated at S240 by Aurora A in mitosis. (A) HeLa cells were blocked at G1/S by double thymidine (Thy.) treatment and then released into fresh medium and harvested at the indicated time point. (B) Mitotic HeLa cell lysates were treated with λ-phosphatase. Samples were analyzed on SDS-PAGE and immunoblotted with the indicated antibodies. (C) HeLa cells were arrested with nocodazole for 17 h and then treated with selected kinase inhibitors (0.25 μM MLN8237 for 30 min, 9 μM RO3306 for 15 min, 0.1 μM AZD1152 for 30 min, 0.1 μM BI2536 for 30 min, respectively). Asy., asynchronized cell samples. The numbers (1.0 and 0.51) indicate the relative gray intensity of p-Aur B. Samples were analyzed on SDS-PAGE and immunoblotted with the indicated antibodies. (D) Mitotic HeLa cells were subjected to an immunoprecipitation (IP) assay with nonspecific IgG or anti-NuSAP antibody followed by Western blotting. (E) Flag-Aurora A was cotransfected with GFP or GFP-NuSAP into HEK293T cells. Lysates of 293T cells were subjected to IP assay with anti-Flag antibody followed by Western blotting. (F) GST-tagged NuSAP protein was incubated with Aurora A kinase at 30°C for 30 min. Samples were processed for MS analysis. Ser-240 site was found to be phosphorylated, consistent with the Aurora A phosphorylation consensus motif. Multiple sequence alignment in the top panel was performed using Uniprot. Red amino acids were conserved. (G) GST-tagged NuSAP proteins with/without point mutation were subjected to Aurora A kinase assay in vitro, followed by autoradiography. GST-tagged MACK was used as positive control. Coomassie Blue staining shows the loading of the GST-tagged NuSAP proteins in the reactions. The red asterisk indicates phosphorylated recombinant NuSAP. Source data are available for this figure: SourceData F2.

NuSAP is phosphorylated at S240 by Aurora A in mitosis. (A) HeLa cells were blocked at G1/S by double thymidine (Thy.) treatment and then released into fresh medium and harvested at the indicated time point. (B) Mitotic HeLa cell lysates were treated with λ-phosphatase. Samples were analyzed on SDS-PAGE and immunoblotted with the indicated antibodies. (C) HeLa cells were arrested with nocodazole for 17 h and then treated with selected kinase inhibitors (0.25 μM MLN8237 for 30 min, 9 μM RO3306 for 15 min, 0.1 μM AZD1152 for 30 min, 0.1 μM BI2536 for 30 min, respectively). Asy., asynchronized cell samples. The numbers (1.0 and 0.51) indicate the relative gray intensity of p-Aur B. Samples were analyzed on SDS-PAGE and immunoblotted with the indicated antibodies. (D) Mitotic HeLa cells were subjected to an immunoprecipitation (IP) assay with nonspecific IgG or anti-NuSAP antibody followed by Western blotting. (E) Flag-Aurora A was cotransfected with GFP or GFP-NuSAP into HEK293T cells. Lysates of 293T cells were subjected to IP assay with anti-Flag antibody followed by Western blotting. (F) GST-tagged NuSAP protein was incubated with Aurora A kinase at 30°C for 30 min. Samples were processed for MS analysis. Ser-240 site was found to be phosphorylated, consistent with the Aurora A phosphorylation consensus motif. Multiple sequence alignment in the top panel was performed using Uniprot. Red amino acids were conserved. (G) GST-tagged NuSAP proteins with/without point mutation were subjected to Aurora A kinase assay in vitro, followed by autoradiography. GST-tagged MACK was used as positive control. Coomassie Blue staining shows the loading of the GST-tagged NuSAP proteins in the reactions. The red asterisk indicates phosphorylated recombinant NuSAP. Source data are available for this figure: SourceData F2.

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