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. 2023 Aug 31;14(8):578. doi: 10.1038/s41419-023-06105-3

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© The Author(s) 2023

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. 5 — A MSpC and MIC cells were transfected with Flag-MIB2 or Flag-circMIB2-P plasmids or silence MIB2 or circMIB2, respectively, and treated with 10 μM CHX. B MSpC cells were transfected with Flag-MIB2 or si-circMIB2 and treated with DMSO or 10 μM MG132 or 10 μM Carfilzomib or 10 μM Bortzeomib. C Flag-MIB2 was co-transfected with GFP-TRAF6 into MSpC cells. Original magnification is 400. D Immunoprecipitation and immunoblot analysis of Flag-MIB2 or Linear-Flag-MIB2-134aa-P in MSpC cells. E Upper panel: Schematic diagram of MIB2-Δ(1-132aa) plasmid construction. Lower panel: Immunoprecipitation and immunoblot analysis of Flag-MIB2, Flag-MIB2-Δ(1-132aa) or Linear-Flag-MIB2-134aa-P, in MSpC cells. F Schematic diagram of TRAF6 and TRAF6 domain mutant plasmid construction. G Immunoprecipitation and immunoblot analysis of Flag-MIB2, with TRAF6 and TRAF6 mutant in MSpC cells. H Immunoprecipitation and immunoblot analysis of Linear-Flag-MIB2-134aa-P with TRAF6 and TRAF6 mutant in MSpC cells. I Coimmunoprecipitation analysis of TRAF6 ubiquitination in MSpC cells transfected HA-ubiquitin-WT in the presence of control vector, Flag-MIB2 or Linear-Flag-MIB2-134aa-P expression plasmid. J Coimmunoprecipitation analysis of TRAF6 ubiquitination in MSpC cells transfected HA-ubiquitin-WT in the presence of control vector, Flag-MIB2, or Flag-MIB2-Δ(1-132aa), or Linear-Flag-MIB2-134aa-P expression plasmid. K Coimmunoprecipitation analysis of TRAF6 ubiquitination in MSpC cells transfected HA-ubiquitin-WT in the presence of control vector, Flag-MIB2, or Flag-MIB2 and Linear-Flag-MIB2-134aa-P expression plasmid. L Immunoprecipitation and immunoblot analysis of Flag-MIB2 or Linear-Flag-MIB2-134aa-P, in MSpC cells. M Coimmunoprecipitation analysis of TRAF6 ubiquitination in MSpC co-transfected with Flag-MIB2 or Linear-Flag-MIB2-134aa-P expression plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11 or HA-ubiqutin-K48 plasmids. All data represented the three independent triplicated experiments.