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. 2024 Jan 15;15:532. doi: 10.1038/s41467-024-44843-w

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© The Author(s) 2024

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. 3 — a Identification of CUL1 as an UBR2-interacting protein by mass spectrometry-based proteomics. Identified CUL1 peptides were shown. b Immunoblotting of CUL1, Flag-tagged UBR2, Myc-tagged (DUSP22), and GAPDH proteins in Jurkat T cells co-transfected with Myc-CUL1, Flag-UBR2, and Myc-DUSP22 plasmids. c Immunoblotting of the endogenous CUL1, Flag-tagged UBR2, Myc-tagged DUSP22, and vinculin proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA plasmids. d CUL1 knockdown inhibited DUSP22-induced UBR2 ubiquitination. Immunoprecipitation and immunoblotting analysis of Lys48-linked ubiquitination of UBR2, Flag-tagged UBR2, Myc-tagged DUSP22, and endogenous CUL1 proteins were performed using the lysates of HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA #3 plasmids. The transfected cells were treated with 25 µM MG132 for 4 h. Arrow, UBR2 protein; asterisk, degraded UBR2 protein. e βTrCP overexpression plus suboptimal DUSP22 (0.8 μg) induced UBR2 degradation. Immunoblotting of Flag-tagged UBR2, Myc-tagged DUSP22, and tubulin proteins in Jurkat T cells co-transfected with Flag-UBR2 and Myc-DUSP22 plus different amounts (0.8 μg, 1.6 μg) of Flag-βTrCP plasmids. f UBR2 interacted with βTrCP. Immunoprecipitation and immunoblotting of Flag-tagged UBR2 with Myc-tagged-βTrCP proteins were performed using the lysates of HEK293T transfected with Flag-UBR2 and Myc-βTrCP plasmids. Anti-vinculin immunoblotting was performed by reprobing the anti-Flag (UBR2) immunoblot membrane. g Confocal microscopy analyses of PLA for the interaction between Flag-tagged UBR2 and Myc-tagged βTrCP proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-βTrCP, and either GFP-DUSP22 or GFP-DUSP22 (C88S) plasmids. Red fluorescence represents the interactions of UBR2 with βTrCP proteins. Images were captured with 400X original magnification. Cell nuclei were stained with DAPI. Scale bar, 50 μm. h Immunoblotting of endogenous UBR2, βTrCP, or CUL1 proteins in Jurkat T cells transfected with scramble shRNAs, βTrCP shRNAs #1, or CUL1 shRNAs #3. T cells were stimulated with anti-CD3 antibody for indicated time periods. i CUL1-βTrCP E3 ligase complex induced UBR2 ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBA1), E2 (top panel, UBE2D3; bottom panel, CDC34), ATP, and SKP1-CUL1-βTrCP-RBX1 complex were co-incubated in the ubiquitination buffer with Flag-tagged UBR2 E3 ligase-inactive mutant (C1210/1213A) proteins.