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. 2017 Nov 7;7:14749. doi: 10.1038/s41598-017-15246-3

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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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Glycine was required in the VEGF signals. (a) HUVECs cultured in the medium with or without amino acids were treated with 20 ng/ml VEGF for 24 hours. Immunoblots of P-S6 and S6 were shown. Membranes were reprobed for ß-actin as loading control. Gray values were analyzed by the Image J software and presented in the graph (n = 3). (b) Intracellular content of alanine(Ala), arginine(Arg), asparagine(Asn), aspartic acid(Asp), cysteine(Cys), γ-aminobutyric acid(Gaba), glutamine(Gln), glutamic acid(Glu), glycine(Gly), histidine (His), isoleucine(Ile), leucine(Leu), lysine 37, methionine(Met), phenylalanine(Phe), proline(Pro), serine(Ser), threonine(Thr), tryptophan(Trp), tyrosine(Tyr) and valine(Val) in control and 20 ng/ml VEGF-treated ECs for 24 h. LC-MS was used to detect relative intracellular quantities of amino acids in ECs in the absence and presence of VEGF (n = 3). (c) Immunoblot of total S6 and P-S6 in untreated and different amino acids-treated ECs. The concentration of amino acids: glycine(140 uM), leu(1.33 mM), tyro(1 mM), ala(1 mM), ser(1 mM). The treatment time was 5 h. Quantitative analysis was presented in the graph (n = 3).