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. 2022 Nov 3;13:6603. doi: 10.1038/s41467-022-34080-4

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© The Author(s) 2022

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 — a Primary Ripk1^+/+ and Ripk1^Y383F/Y383F BMDMs were stimulated with TNF for indicated time points. Whole-cell lysates were immunoprecipitated with anti-phospho-tyrosine antibody for western blotting with indicated antibodies. b, c Ripk1^+/+ and Ripk1^Y383F/Y383F immortalized MEFs were stimulated by TNF (10 ng/ml) for indicated time points. Cytosolic (b) and nuclear (c) extractions were collected for western blotting with indicated antibodies. d Primary Ripk1^+/+ and Ripk1^Y383F/Y383F BMDMs were stimulated with TNF at different time point. The transcriptional and expression level of inflammatory NF-κB target genes were measured by qPCR. Ripk1^+/+ and Ripk1^Y383F/Y383F immortalized MEFs were stimulated by Flag-TNF (100 ng/ml) (e) or TNF (10 ng/ml) /BV-6 (2.5 μM) (f) for indicated time points. Whole cell lysates were immunoprecipitated by anti-Flag resin (e) or anti-RIPK1 antibodies (f) for western blotting with indicated antibodies. Ripk1^+/+ and Ripk1^Y383F/Y383F immortalized MEFs with or without knockdown of MK2 (g, h) or overexpression of constitutively active MK2 (MK2-CA) (i, j) were treated with TNF (10 ng/ml) /BV-6 (2.5 μM) for indicated time points. Whole-cell lysates were collected for western blotting (g, i). Cell death was measured by SytoxGreen positivity (h, j). T: TNF (10 ng/ml); B: BV-6 (2.5 μM); Z: zVAD.fmk (20 μM). In d, h, j, data are represented as mean ± SEM (n = 3 independent cell samples). Statistical significance was determined using a two-tailed unpaired t test. n.s. p > 0.05; ** p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided with this paper.