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. 2017 Nov 9;7:15172. doi: 10.1038/s41598-017-15173-3

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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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IL-12-stimulation induced intracellular signaling of synthetic receptors mimicking the IL-35 receptor complex IL-12Rβ2:WSX-1. (A) Schematic overview of IL-35-type signaling by IL-12-induced receptor activation of IL-12Rβ1_EXR-WSX-1_IR and IL-12Rβ2. (B) Representative histograms of IL-12Rβ1_EXR-WSX-1_IR (upper panel) and IL-12Rβ2 (lower panel) surface expression of Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR/IL-12Rβ2 cells (light solid lines). Gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (C) Analysis of STAT1/3 and Erk1/2 activation. Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR and Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR/IL-12Rβ2 cells were washed three times, starved, and stimulated with 4 ng/ml HIL-12 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT1/3/Erk1/2 and STAT1/3/Erk1/2. Western blot data show one representative experiment out of three. (D) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR and Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR/IL-12Rβ2 cells. Equal numbers of cells were cultured for 3 days in the presence of 4 ng/ml HIL-12. Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6–induced proliferation (10 ng/ml) was set to 100%. One representative experiment out of three is shown. Error bars represent SD. Statistical analysis used a Welch t test (n = 3; ns = not significant; ***p ≤ 0.001). (E) Cellular proliferation of Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR/IL-12Rβ2 and Ba/F3-gp130/IL-12Rβ1/IL-12Rβ2 cells. Equal numbers of cells were cultured for 3 days in the presence of HIL-12 (0.01 to 500 ng/ml). Proliferation was measured using the colorimetric CellTiter-Blue Cell Viability Assay. HIL-6–induced proliferation (10 ng/ml) was set to 100%. One representative experiment out of four is shown. Error bars represent SD. (F) Analysis of STAT3 target gene expression of Pim-1 in Ba/F3-gp130/IL-12Rβ1_EXR-WSX-1_IR/IL-12Rβ2 cells stimulated with 4 ng/ml IL-12 for 2 h. One representative experiment out of two is shown.