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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 interacts with NLRP3. (A) Flag-NLRP3 was cotransfected with GFP-TRIM65 in HEK-293T cells. The interaction between NLRP3 and TRIM65 was analyzed by immunoprecipitation and western blotting. (B) Flag-NLRP3 was transfected and expressed in HEK-293T cells. The interaction between NLRP3 and endogenous TRIM65 was assayed by immunoprecipitation and western blotting. (C) The lysates of PMA-differentiated and LPS-primed THP-1 cells were immunoprecipitated with anti-NLRP3 antibody, and the interaction between endogenous NLRP3 and endogenous TRIM65 was analyzed by western blotting. (D) mCherry-NLRP3 was cotransfected with GFP-TRIM65 in HEK-293T cells. The colocalization of NLRP3 and TRIM65 was assayed by confocal microscopy. Bars: (white) 20 mm. (E) Diagram of full-length TRIM65 and TRIM65 deletion mutants (ΔRING, ΔR+ΔBB, ΔCC+ΔS or ΔSPRY). (F) Flag-TRIM65 and TRIM65 deletion mutants were individually cotransfected with VSV-NLRP3 in HEK-293T cells. The interactions between NLRP3 and TRIM65 or its different domains were analyzed by immunoprecipitation and western blotting. (G) Full-length or truncated Flag-NLRP3 (Flag-PYD, Flag-NACHT or Flag-LRR) was cotransfected with GFP-TRIM65 in HEK-293T cells. The interaction between TRIM65 and full-length or truncated NLRP3 was analyzed by immunoprecipitation and western blotting. Data are representative of three independent experiments. EV, empty vector; Input, cell extract without immunoprecipitation.