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. 2020 Jul 9;10:11388. doi: 10.1038/s41598-020-68219-4

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© The Author(s) 2020

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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MK2-mediated phosphorylation of SRC-3 at S857 in response to TNF-α is dose and time dependent. (A) TNF-α stimulation phosphorylates SRC-3 and MK2 in a dose dependent manner. MDA MB 231 cells were seeded and left in incubator overnight. After that, the cells were treated with 0, 5, 10, 20 or 30 ng/ml of TNF-α as indicated in the figure for 15 min. Then the cells were lysed and the phosphorylation statuses of SRC-3 at S857 and MK2 at threonine (T) 334 as well as total protein amounts of these proteins and actin were examined by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-MK2, anti-MK2 and anti-actin antibodies. *indicates the phosphorylated band. The full-length blots are presented in supplementary figure S30. (B, C) TNF-α stimulation causes phosphorylation of SRC-3 at S857 and MK2 in a time dependent manner. MDA MB 231 (B) or A549 (C) wild type cells were seeded and left overnight. Then the cells were treated with 10 ng/ml TNF-α for 0, 15, 30, 60, 120, 240 or 360 min as indicated in the figures. The cells were lysed and phosphorylation status was examined by Western-blotting as described in (A) above. *indicates the phosphorylated band. The full-length blots are presented in supplementary figures S31,S32.