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. 2016 Apr 19;7:8. doi: 10.1186/s13100-016-0064-x

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© Kines et al. 2016

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Fig. 6 — Chronic expression of EN proteins containing mutations in putative phosphorylation sites causes toxicity. HeLa cells were transfected with a single expression plasmid containing both Hygro^R and the indicated EN putative phosphorylation mutant sequence. Hygromycin selection was maintained for 2 weeks post-transfection, allowing stable expression of ENp throughout the assay. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control indicates cells transfected with an empty vector. Colony formation was assayed after 2 weeks under hygromycin (Y-axis) and used as a measure of toxicity as previously described [27, 43]. Asterisks indicate a statistically significant difference in the relative number of Hygro^R colonies compared to EN (t-test, P ≤ 0.05)