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. 2016 Aug 24;291(40):21123–21136. doi: 10.1074/jbc.M116.745372

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Phosphorylation of Sds22 promotes Aurora B kinase activity. A, phosphorylation of a kinetochore Aurora B substrate CENP-A is decreased by expression of Sds22^5A. Compared with Sds22^5A-GFP, overexpression of Sds22^WT-GFP and Sds22^5D-GFP produces stronger CENP-A Ser(P)⁷ staining on the kinetochores. Scale bar, 10 μm. B, statistical analyses of CENP-A Ser(P)⁷ immunofluorescence intensity at the kinetochores as in A indicate that Sds22^5A decreases Aurora B activity. Error bars represent means ± S.E.; >20 cells of each categories from three independent preparations. Student's t test was used to calculate p value for comparison. *, p < 0.05; **, p < 0.01. C, phosphorylation of CENP-A at Ser⁷ (a kinetochore substrate of Aurora B) is increased by Sds22^5D expression in PLK1-depleted HeLa cells. Compared with Sds22^5D-GFP, overexpression of Sds22^WT-GFP and Sds22^5A-GFP produces weaker CENP-A Ser(P)⁷ staining on the kinetochores. Scale bar, 10 μm. D, statistical analyses of CENP-A Ser(P)⁷ immunofluorescence intensity at the kinetochores as in C indicatethat Sds22^5D expression increases Aurora B activity in PLK1-depleted HeLa cells. Error bars represent means ± S.E.; >20 cells of each categories from three different independent preparations. Student's t test was used to calculate p value for comparison. *, p < 0.05; ***, p < 0.001. E, the expression levels of Sds22^WT-GFP and its mutants in endogenous PLK1-depleted HeLa cells. Cells were collected at 48 h after transfection and analyzed by Western blotting analyses using indicated antibodies. Tubulin served as loading control. F, phosphorylation of CENP-A at Ser⁷ is decreased by expression of Sds22^5A in Sds22-depleted HeLa cells. Compared with Sds22^5A-GFP, in Sds22 depleted HeLa cells, overexpression of Sds22^WT-GFP and Sds22^5D-GFP produces stronger CENP-A Ser(P)⁷ staining on the kinetochores. Scale bar, 10 μm. G, statistical analyses of CENP-A Ser(P)⁷ immunofluorescence intensity at the kinetochores as in F indicate that in Sds22-depleted HeLa cells, Sds22^5A decreases Aurora B activity (Error bars represent means ± S.E.; >20 cells of each categories from three different preparations). Student's t test was used to calculate p value for comparison. **, p < 0.01; ***, p < 0.001. H, phosphorylation of CENP-A at Ser⁷ is decreased by kinetochore targeted Sds22^5A in Sds22-depleted HeLa cells. Compared with Hec1-mCherry-Sds22^5A, in Sds22-depleted HeLa cells, overexpression of Hec1-mCherry-Sds22^WT and Hec1-mCherry-Sds22^5D produced stronger CENP-A Ser(P)⁷ staining on the kinetochores. Scale bar, 10 μm. I, statistical analyses of CENP-A Ser(P)⁷ immunofluorescence intensity at the kinetochores as in H indicate that in Sds22-depleted HeLa cells, kinetochore targeted Sds22^5A decreases Aurora B activity. Error bars represent means ± S.E.; >20 cells of each categories from three different preparations. Student's t test was used to calculate p value for comparison. ***, p < 0.001. J, the expression levels of Hec1-mCherry-Sds22^WT and its mutants in endogenous Sds22-depleted HeLa cells. Cells were collected at 48 h after transfection and analyzed by Western blots using indicated antibodies. Tubulin served as loading control. ACA, anti-centromere antibody.