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. 2023 Feb 2;21:29. doi: 10.1186/s12964-022-01022-y

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© The Author(s) 2023

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 5 — NEDD4 indirectly promotes proteasomal degradation of NLRP3. A, B Immunoblot analysis and quantification by densitometry of NLRP3, NLRC4, AIM2, GSDMD and C-GSDMD protein or RT-PCR analysis of mouse peritoneal macrophages from NEDD4^+/+ or NEDD4^−/− mouse, and then stimulated with LPS/Nig at different times. Data are expressed as mean ± SD (n = 3). *P < 0.05 vs. NEDD4+/+group. C, D Immunoblot analysis and quantification by densitometry of NLRP3 from THP-1 cells silenced of NEDD4, and then treated for various times with cycloheximide (CHX). Data are expressed as mean ± SD (n = 3). *P < 0.05 vs. ctrl siRNA group. E, F Immunoblot analysis of extracts from THP-1 cells stimulated with LPS for 4 h, and followed by stimulation with Nig for 1 h. Then treated with MG-132 and chloroquine (CQ) for 4 h. Data are expressed as mean ± SD (n = 3). *P < 0.05 vs. LPS+Nig, **P < 0.05 vs. LPS+Nig+MG132, ***P < 0.05 vs. LPS+Nig+CQ. G Immunoblot analysis of extracts from HEK293T cells transfected with Myc-NLRP3 and Flag-NEDD4 expression plasmid then treated with 3-MA. H HEK293T cells were transfected with NEDD4 and NLRP3, and cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibody. The immunocomplex was analyzed by immunoblotting. I CMECs transfected with GFP-NEDD4 and RFC-NLRP3 were fixed and incubated with a secondary antibody conjugated to Alexa Fluor 594 and Alexa Fluor 488. Non-colocalization between NEDD4 and NLRP3 was examined by fluorescence microscope