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. 2019 Sep 13;10:4157. doi: 10.1038/s41467-019-12033-8

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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. 3 — Ripk1^K376R/K376R mutation increases RIPK1 kinase activity. a Immortalized Ripk1^+/+, Ripk1^K376R/K376R, and Ripk1^−/− MEFs were treated for the indicated time with TNFα (20 ng/ml). The K63-ubiquitylated proteins were isolated by K63-TUBEs and analyzed by western blot. b TNF-RSC was immunoprecipitated using anti-Flag resin in Ripk1^+/+ and Ripk1^K376R/K376R immortalized MEFs treated for the indicated time with Flag-TNFα (100 ng/ml). The immunocomplexes were analyzed by western blot with indicated antibodies. c Nuclear extracts were collected from Ripk1^+/+, Ripk1^K376R/K376R, and Ripk1^−/− immortalized MEFs treated with TNFα (20 ng/ml) at indicated times and analyzed by western blotting with antibodies against p65 and PCNA. d Ripk1^+/+ and Ripk1^K376R/K376R immortalized MEFs that stably expressed with the Flag-tagged IκBα-SR were stimulated with TNFα (40 ng/ml) for different periods of time. Cell death was measured by SytoxGreen positivity. e, f Complex II was immunoprecipitated using RIPK1 antibody (e) or RIPK3 antibody (f) in Ripk1^+/+, Ripk1^K376R/K376R, and Ripk1^−/− immortalized MEFs treated for the indicated time with TSZ (e) or TZ (f). The immunocomplexes were analyzed by western blotting with indicated antibodies. T, TNFα (20 ng/ml); S, Smac mimetics (1 μM); Z, zVAD.fmk (1 μM). g Ripk1^+/+ and Ripk1^K376R/K376R immortalized MEFs that expressed with the Flag-tagged TAK1/TAB1 or TAK1-∆N were stimulated with TNFα (20 ng/ml) plus zVAD.fmk (10 μM) for different periods of time. The cell lysates were analyzed by western blotting with indicated antibodies. h Cell death of Ripk1^+/+ and Ripk1^K376R/K376R immortalized MEFs treated for 5 h with different stimulators was measured by SytoxGreen positivity. T: TNFα (20 ng/ml), Z: zVAD.fmk (10 μM), TAK1i: TAK1 inhibitor (500 μM), IKKi: IKK inhibitor(5 μM), MK2i: MK2 inhibitor (2 μM). In d, h, data are mean ± s.e.m. (n = 3 independent cell samples for each genotype). Statistical significance was determined using a two-tailed unpaired t test, n.s., P > 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001