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. 2017 Nov 10;7:15262. doi: 10.1038/s41598-017-15509-z

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Amniotic membrane (AM) promotes FAK activation and detection at focal structures in migrating HaCaT cells. (a) HaCaT cells at the migrating edge of artificial wound assays show increased p-tyr-100 immune-staining when AM was present. P-tyr-100 immune-staining signal was converted into pseudo-color rainbow scale to display fluorescence-intensity variations. Fluorescent signal was converted using Rainbow feature from ZEN software, on a linear mode and covering the full range of the data. Co-staining with phalloidin and Hoechst-33258 was used to show the cell structure and nuclei, respectively. P-tyr-100: pseudo-color; p-tyr-100: green; Actin: red; Nuclei: blue. Scale Bar 25 µm. (b) Relative fluorescence level plot generated from average p-tyr-100 signal intensity data obtained at the migrating leading edge. (c) Western Blot of total protein extracts from sub-confluent HaCaT cells cultured in the presence of AM and/or different inhibitors collected for a 24 hours time course. ß-actin was used a loading control. Relative protein level plots were generated from Western Blot quantification data. C: serum starvation; JNKi: SP600125; MEKi: PD98059. Asterisks denote statistically significant differences between conditions according to ANOVA statistical analysis: (***) p < 0.001; (ns) not significant. All the experiments were repeated at least three times. Representative pictures and results are shown.