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. 2019 Jan 18;2:27. doi: 10.1038/s42003-018-0259-4

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© The Author(s) 2019

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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Fig. 4 — The SOCS3-negative feedback loop increases robustness of STAT3 activation against varying STAT3 expression. a MEF, MEF STAT3^high, and MEF SOCS3^-/- cells were stimulated with 75 ng Hy-IL-6 per ml for the indicated times. SOCS3, and HSC70 protein expression were evaluated by Western blotting. A representative result of n = 6 independent experiments is shown. Uncropped Western blots are depicted in Supplementary Figure 7a, b. b MEF, MEF STAT3^high, and MEF SOCS3^-/- cells were stimulated with 50 ng Hy-IL-6 per ml or with 75 ng Hy-IL-6 per ml for 15 and 90 min or left untreated. STAT3 phosphorylation was evaluated by intracellular flow cytometry. For independent experiments mean fluorescence of cells per time point was calculated. Maximal mean fluorescence in each experiment was normalised to 100 %. Data from n = 3 experiments. c MEF SOCS3^-/- cells were stimulated with 75 ng Hy-IL-6 per ml for the indicated times. STAT3 phosphorylation was evaluated by intracellular flow cytometry. d MEF SOCS3^-/- cells were stimulated with increasing amount of Hy-IL-6 for 15 min. STAT3 phosphorylation and expression were evaluated by intracellular multiplex flow cytometry using specific fluorescent antibodies against STAT3 (pY)705 and STAT3 (Supplementary Figure 8b). For independent experiments, mean fluorescence of cells per cytokine dose was calculated. Maximal mean fluorescence in each experiment was normalised to 100%. The data are from n = 4 experiments. e Based on Fig. 4d and Supplementary Figure 8b, Mutual Information between STAT3 expression and STAT3 phosphorylation in MEF SOCS3^-/- cells stimulated for 15 min with Hy-IL-6 was calculated. The data are from n = 4 independent experiments. Overlay with Fig. 2g (MEF) for direct comparison of MEF cells and MEF SOCS3^-/- cells. f Based on the data presented in Supplementary Figure 9 (for MEF), S10 (for MEF SOCS3^-/-), and S11 (for MEF STAT3^high), Mutual Information between STAT3 expression and IL-6-induced STAT3 phosphorylation in MEF (light grey), MEF SOCS3^-/- (white) and MEF STAT3^high (dark grey) cells stimulated for 90 min with Hy-IL-6 was calculated. The data are from n = 3, 4, and 4 independent experiments, respectively