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. 2018 Apr 19;9:1560. doi: 10.1038/s41467-018-03669-z

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© The Author(s) 2018

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. 7 — Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples (a) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates (b) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates (c) from WT and Peli2^−/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2^−/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples (e) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates (f) from WT and Peli2^−/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2^−/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. *p < 0.05 (paired, two-tailed Student’s t-test). NS, not significant. Data are biological replicates that are representative of three independent experiments (a-c, e-g) or mean ± s.e.m. of three independent experiments (d)