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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. 5 — STAT3-S727 phosphorylation increases robustness of STAT3 phosphorylation against varying STAT expression. a STAT3 expression in MEF, MEF STAT3^high, and MEF STAT3-S727A^high cells was evaluated by flow cytometry using fluorescent antibodies against STAT3. For independent experiments mean fluorescence of cells per cell line was calculated. Mean fluorescence of STAT3 expression in MEF STAT3^high cells was set to 100%. The data are from n = 3 experiments. b MEF STAT3^high and MEF STAT3-S727A^high cells were stimulated with 75 ng Hy-IL-6 per ml for the indicated times. STAT3-S727 phosphorylation and HSC70 expression were evaluated by Western blotting. A representative result of n = 6 independent experiments is shown. Uncropped Western Blots are shown in Supplementary Figure 12a, b. c MEF STAT3-S727A^high cells were stimulated with 75 ng Hy-IL-6 per ml for the indicated times. STAT3 phosphorylation was evaluated by flow cytometry. d MEF STAT3-S727A^high cells were stimulated with increasing amount of Hy-IL-6 for 15 min. STAT3 phosphorylation and expression were evaluated by flow cytometry using fluorescent antibodies against STAT3 (pY)705 and STAT3 (Supplementary Figure 13). The data are pooled from n = 3 experiments. e MEF STAT3-S727A^high cells were stimulated with increasing amount of Hy-IL-6 for 90 min and analysed as in d) (Supplementary Figure 14). For independent experiments, mean fluorescence of cells per cytokine dose was calculated. Maximal mean fluorescence in each experiment was normalised to 100 %. Data are from n = 3 experiments. f Based on data presented in Fig. 5d and Supplementary Figure 13, or Fig. 5e and Supplementary Figure 14, Mutual Information between STAT3 expression and IL-6-induced STAT3 phosphorylation in MEF STAT3-S727A^high cells stimulated with Hy-IL-6 for 15 min (white) or 90 min (black) was calculated. Data are from n = 3 independent experiments. g Overlay of Fig. 3d and Fig. 3f. h Overlay of Fig. 4f and Fig. 5f. i MEF, MEF STAT3^high, and MEF STAT3-S727A^high cells were stimulated with 100 ng Hy-IL-6 per ml for 15 min. STAT3 phosphorylation in MEF, MEF STAT3^high, and MEF STAT3-S727A^high cells was evaluated by flow cytometry. For independent experiments mean fluorescence of cells per cell line was calculated. Mean fluorescence of STAT3 phosphorylation in MEF STAT3^high cells was set to 100%. The data are from n = 3 experiments