Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2001 Dec 24;155(7):1147–1158. doi: 10.1083/jcb.200108125

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2001, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 2. — Aurora B phosphorylates the NH ₂ termini of histone H3 and CENP-A in vitro. Arrows indicate the migration of CENP-A–GST. (a) Immunoprecipitated Aurora B phosphorylates both H3 and CENP-A NH₂ termini expressed as GST fusions. Top, representative Coomassie stain of SDS-PAGE. Lower bands are GST alone or fused to the NH₂ terminus of either H3 or CENP-A; upper bands are antibody light chain, either anti–Aim-1 or control isotype-matched mouse IgG1. Also shown are mock reactions where GST fusions were mixed with the protein G sepharose beads used in immunoprecipitation. Bottom, autoradiogram of the same gel. Aurora B clearly phosphorylates the H3 and CENP-A NH₂ termini. No signal is seen in either of the negative controls when the Aurora B antibody is not present. (b) Aurora B immunoprecipitation significantly concentrates the Aurora B kinase activity. Whole extract mixed with GST fusions does not result in phosphorylation of CENP-A or H3 NH₂ termini (extract), and the Aurora B antibody alone contains no kinase activity (antibody). Whole extract alone allowed to autophosphorylate does not reveal CENP-A or H3 bands (auto). (c) Ser7 is a target Aurora B phosphorylation site in the CENP-A NH₂ terminus. Aurora B immunoprecipitations were mixed with the CENP-A NH₂-terminal GST fusion (WT), or site-directed mutants of this construct (S7A and S7E). CENP-A phosphorylation is reduced 50% by mutation of Ser7; however, it is not abolished. Immunoprecipitation with an irrelevant antibody (mouse IgG1) is negative. (d) Mutation of CENP-A Ser7 abolishes reactivity with the anti–CENP-A-Ser7P antibody. GST fusion proteins were incubated with immunoprecipitated Aurora B in the presence of cold ATP before Western blotting. Top, ponceau stain; bottom, Western blot. Only wild-type CENP-A reacts positively with the Ser7P antibody. There is no cross-reaction with GST or the NH₂ terminus of H3, and mutation of Ser7 to alanine (S7A) or glutamate (S7E; unpublished data) abolishes reactivity.