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. 2021 Aug 26;12:741839. doi: 10.3389/fimmu.2021.741839

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Copyright © 2021 Tang, Li, Zhou, Wang, Fan, Yang and Qi

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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TRIM65 promotes NLRP3 lys48- and lys63- linked ubiquitination. (A) Flag-NLRP3 and GFP-TRIM65 were cotransfected with HA-Ubiquitin, HA-Ubiquitin (K48-linked only), or HA-Ubiquitin (K63-linked only) in HEK-293T cells. The ubiquitination of NLRP3 was analyzed by immunoprecipitation and western blotting. (B, C) Flag-NLRP3 was cotransfected with GFP-TRIM65 in HEK- 293T cells. The overall ubiquitination (B), K48-linked ubiquitination (C) and K63-linked ubiquitination (C) of NLRP3 were analyzed by immunoprecipitation and western blotting. (D) Flag-NLRP3 was cotransfected with GFP-TRIM65 or GFP-TRIM65 lacking the RING domain in HEK-293T cells. The ubiquitination of NLRP3 was analyzed by immunoprecipitation and western blotting. (E, F) Full-length Flag-NLRP3 and NLRP3 truncation mutants were individually cotransfected with GFP-TRIM65 in HEK-293T cells. Ubiquitination of NLRP3 or its truncation mutants was analyzed by immunoprecipitation and western blotting. (G) Endogenous ubiquitination of NLRP3 in PMA-differentiated and LPS-primed THP-1 cells stably expressing scrambled shRNA or shRNA targeting TRIM65 mRNA was analyzed by immunoprecipitation and western blotting. (H) LPS-primed BMDMs from wild-type or Trim65^-/- mice were stimulated with nigericin, and the endogenous ubiquitination of NLRP3 was analyzed by immunoprecipitation and western blotting. Data are representative of two or three independent experiments. EV, empty vector.