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. 2008 May 13;105(20):7153–7158. doi: 10.1073/pnas.0710018105

Fig. 3.

Fig. 3.

EB1 is not a substrate of Aurora-B and knockdown of its expression impairs Aurora-B kinase activity. (A) Aurora-B kinase assays were performed on HA-Aurora-B immunoprecipitate (AuB IP) or purified His-Aurora-B (AuB), using GST, GST-EB1, or GST-histone H3 as the substrate. The reaction mixture was subjected to SDS/PAGE and analyzed by autoradiography. The levels of GST-fusion proteins used in the kinase assays were examined by Coomassie blue staining. (B) Western blots showing the decrease of EB1 expression by three different siRNAs, including two targeting the coding sequence (E-1 and E-2) and one targeting the untranslated region (E-3). (C) HeLa cells transfected with control or EB1 siRNAs were examined by immunofluorescence microscopy with a phospho-H3 (serine-10)-specific antibody. Total cell population was detected by staining cells with DAPI. (D) Cells were transfected with the indicated siRNAs and pCMV-HA-Aurora-A, pCMV-HA-Aurora-B, or pCMV-HA-Aurora-C. Kinase assays were then performed on HA-Aurora-A, HA-Aurora-B, or HA-Aurora-C immunoprecipitate, using H3 as the substrate. The reaction mixtures were subjected to SDS/PAGE, and H3 phosphorylation was analyzed by autoradiography. The levels of HA-Aurora-B immunoprecipitate used in the kinase assays were examined by Western blotting with anti-HA antibody. (E) Cells were transfected with control siRNA or EB1 siRNA (E-3) and pCMV-HA-Aurora-B, together with GST or GST-EB1. Kinase assays were performed on HA-Aurora-B immunoprecipitate as in D. The levels of HA-Aurora-B immunoprecipitate used in the kinase assays were examined by Western blotting.