Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are
encouraged to use the Version of Record that, when published, will replace this version. The most
up-to-date version is available at http://dx.doi.org/10.1042/BCJ20160964. Please cite using the DOI
10.1042/BCJ20160964
BIOCHEMICAL JOURNAL
ACCEPTED MANUSCRIPT
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Cite as Biochemical Journal (2017) DOI: 10.1042/BCJ20160964
Copyright 2017 The Author(s).
Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under which the
article is published. Archiving of non-open access articles is permitted in accordance with the Archiving Policy of Portland Press
(http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).
Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under
which the article is published. Archiving of non-open access articles is permitted in accordance with the
Archiving Policy of Portland Press (
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ACCEPTED MANUSCRIPT
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