Figure 4. EB1 is in close proximity to... | Rockefeller University Press

Figure 4.

$Figure 4. EB1 is in close proximity to Aurora B at centromeres. (A) Microtubules stimulate Aurora B bound to INCENP790–856 (AI790–856) activity on MBP in vitro. The assay was performed with or without 3 µM taxol-stabilized microtubules (MT). (B) EB1 does not stimulate Aurora B kinase activity alone or in combination with microtubules. AI790–-856 in vitro kinase assay, using MBP as a substrate, showing the effect of adding 3 µM taxol-stabilized microtubules and 100 ng EB1 separately or in combination. Kinase activity of 20 nM AI790–856 was assayed similarly as in A. (C) Direct interaction between EB1 and the catalytic subunit of the CPC. Recombinant Xenopus GST–Aurora B bound to a fragment of INCENP790–856 (GST-AI) on glutathione–Sepharose 4 beads was incubated with the indicated concentration of recombinant xEB1, washed, and eluted with glutathione, and the two peak fractions of the elutions (E1 and E2) were quantified by immunoblotting. GST beads were used as a control. Molecular markers are given in kilodaltons. (D) Immunostaining of EB1 and Borealin in HeLa cells. (The single channel split of images is shown in Fig. S4 A.) Bar, 2.6 µm. (E) HeLa cells immunostained for Borealin and tubulin and the close proximity of EB1 and Aurora B shown by PLA. Insets illustrate PLA at individual centromeres. Prometaphase inset is a projection of four z sections, and metaphase inset is a projection of seven z sections (PLA controls are shown in Fig. S4, C and C′). Bars: (main images) 2.4 µm; (prometaphase inset) 0.39 µm; (metaphase inset) 0.35 µm. (F) Quantification showing percentage of occurrence of PLA spots proximal to centromeres from the PLA experiment shown in C. Error bars show standard deviations.$

EB1 is in close proximity to Aurora B at centromeres. (A) Microtubules stimulate Aurora B bound to INCENP^790–856 (AI^790–856) activity on MBP in vitro. The assay was performed with or without 3 µM taxol-stabilized microtubules (MT). (B) EB1 does not stimulate Aurora B kinase activity alone or in combination with microtubules. AI^790–-856 in vitro kinase assay, using MBP as a substrate, showing the effect of adding 3 µM taxol-stabilized microtubules and 100 ng EB1 separately or in combination. Kinase activity of 20 nM AI^790–856 was assayed similarly as in A. (C) Direct interaction between EB1 and the catalytic subunit of the CPC. Recombinant Xenopus GST–Aurora B bound to a fragment of INCENP^790–856 (GST-AI) on glutathione–Sepharose 4 beads was incubated with the indicated concentration of recombinant xEB1, washed, and eluted with glutathione, and the two peak fractions of the elutions (E1 and E2) were quantified by immunoblotting. GST beads were used as a control. Molecular markers are given in kilodaltons. (D) Immunostaining of EB1 and Borealin in HeLa cells. (The single channel split of images is shown in Fig. S4 A.) Bar, 2.6 µm. (E) HeLa cells immunostained for Borealin and tubulin and the close proximity of EB1 and Aurora B shown by PLA. Insets illustrate PLA at individual centromeres. Prometaphase inset is a projection of four z sections, and metaphase inset is a projection of seven z sections (PLA controls are shown in Fig. S4, C and C′). Bars: (main images) 2.4 µm; (prometaphase inset) 0.39 µm; (metaphase inset) 0.35 µm. (F) Quantification showing percentage of occurrence of PLA spots proximal to centromeres from the PLA experiment shown in C. Error bars show standard deviations.