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. 2021 May 11;12:2674. doi: 10.1038/s41467-021-22987-3

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© The Author(s) 2021

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 — a Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged K27-linked ubiquitin (K27-Ub), β-TrCP1 and Myc-NLRP3 or indicated mutant Myc-NLRP3, followed by IP with anti-Myc, probed with anti-HA. b Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, Myc-NLRP3 K380R, and then treated for various times with cycloheximide (CHX, 100 ug/mL) (top). NLRP3 and NLRP3 K380R expression levels were quantitated by measuring band intensities using “ImageJ” software. The values were normalized to actin (bottom) (mean ± SD, two-way ANOVA with Bonferroni test, NLRP3-K380R vs. NLRP3-WT lower panel: *P = 0.0129, *P = 0.0321, **P = 0.0010 in sequence; n = 3 independent experiments). c Immunoblot analysis of lysates from HEK293T cells transfected with Flag-β-TrCP1 and Myc-NLRP3 or Myc-NLRP3 K380R. d ELISA of IL-1β in supernatants from HEK293T cells transfected with ASC, pro-caspase-1, pro-IL-1β, NLRP3 or NLRP3 K380R plasmids and stimulated with 10 μM nigericin (mean ± SD, one-way ANOVA with Bonferroni test, the third group vs. second group ***P < 0.0001; n = 3 independent experiments). e, f NLRP3^−/− iBMDMs were infected with lentivirus expressing mouse WT or NLRP3-K380R, respectively. Immunoblot analysis of indicated proteins (e), then treated these cells with LPS plus ATP or nigericin for detection of IL-1β and TNF-α in supernatants (f) (mean ± SD, two-way ANOVA with Bonferroni test, NLRP3-K380R vs. NLRP3-WT left panel: *P = 0.0475, **P = 0.0037 in sequence, n = 3 independent experiments). Similar results were obtained from three independent experiments. Source data are provided as a Source Data file.