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. 2020 Nov 20;11:5919. doi: 10.1038/s41467-020-19670-4

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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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Fig. 4 — Cells in all panels are Atg16L1^−/− MEFs engineered to harbor a STAT3-luciferase reporter system and to express STAT3, subsequently restored with full-length ATG16L1 (FL) or a deleted version lacking the WDD (Nt, 1–299), and retrovirally transduced to express IL-10Rs A and B. a Co-precipitation between IL-10RB and ATG16L1 as a function of IL-10 treatment. The indicated cells (FL or Nt) were treated with IL-10 and E64d/pepstatin (10 μg/ml each) for 30 min or left untreated. Cells were then lysed and processed for anti-Flag immunoprecipitation. Shown are Western-blots against the indicated molecules. IP: immunoprecipitate; TL: total lysate. b Time-course and dose-response activation of STAT3-luciferase activity by IL-10. The indicated cells (FL or Nt) were treated with IL-10 for the shown times (100 ng/ml; top panel) or with the indicated doses of IL-10 (4 h; bottom panel), and lysed to measure luciferase activity. Data are expressed as the mean of fold induction of luciferase activity −/+ s.d of triplicate experimental points (n = 3; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-sided Student’s t-test) from a representative experiment. c Pulse-chase assay of IL-10-induced STAT3 phosphorylation. Cells (F: FL, N: Nt) were pre-incubated with IL-10 (50 ng/ml; 30 min on ice), incubated at 37 °C for the shown times and lysed to obtain cytoplasmic and nuclear extracts for Western-blotting against the indicated molecules (H3, Histone-3). Corrected densitometric quantifications are shown at the bottom of the Western-blot images. d Immunofluorescence assay to evaluate the number of Flag-positive intracellular vesicles in IL-10 treated cells. The indicated cells were incubated with IL-10 (50 ng/ml) for the shown times and processed for anti-Flag immunofluorescence. Shown are representative confocal pictures (left). The graph (right) displays the number of Flag-positive vesicles per cell in the different conditions. Data are presented as box-plots where the central line represents the median value, the box shows percentiles 25–75 and the whiskers include the most extreme values (n = 56, n = 68 cells for untreated and treated samples, respectively; *P < 0.05; ****P < 0.0001, two-sided Student’s t-test).