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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. 6 — a Immunoprecipitations of the endogenous Lck with either UBR2 or DUSP22 proteins in the lysates of anti-CD3-stimulated murine primary T cells. b TCR signaling stimulated Lys63-linked ubiquitination and Tyr394 phosphorylation of Lck in murine primary T cells. Endogenous Lck immunocomplexes in the lysates of anti-CD3-stimulated T cells were immunoprecipitated with anti-Lck antibody and then subjected to immunoblotting. c Confocal microscopy analyses of PLA for the ubiquitinated and phosphorylated Lck in TCR-stimulated T cells of wild-type (WT) or UBR2 knockout mice using anti-Lck antibody plus either anti-ubiquitin (Lys63) or anti-phospho-Lck (Tyr394) antibodies. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. d UBR2 induced Lck ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBE1), E2 (UBE2D3), Flag-Lck, and ATP were co-incubated in the ubiquitination buffer with E3 (Flag-tagged UBR2 or Flag-tagged UBR2 ligase-inactive mutant (C1210/1213A)) proteins. e The Lys99 and Lys276 residues were identified as ubiquitination sites of Lck by mass spectrometry analysis. K(ub), ubiquitinated lysine residue. f Immunoprecipitations of ubiquitinated Flag-tagged Lck with Myc-tagged UBR2 proteins in the lysates of indicated HEK293T transfectants. Flag-tagged Lck proteins were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-ubiquitin (Lys63) antibody or anti-Flag antibody. g Immunoprecipitation and immunoblotting analysis of Flag-tagged Lck with Tyr394 phosphorylated Lck proteins in the lysates of Jurkat T cells transfected with Flag-Lck (WT) or Flag-Lck (K99/276R) plasmid. h Confocal microscopy analyses of PLA for the ubiquitinated Lck and Tyr394-phosphorylated Lck in TCR-stimulated Jurkat transfectants using anti-Lck plus anti-ubiquitin (Lys63) and anti-phospho-Lck (Tyr394) antibodies, respectively. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. i Double mutations (K99/276R) of Lck inhibited phosphorylation of Lck at Tyr394 residue by in vitro kinase assay. j In vitro ubiquitin E3 ligase assay in combination with kinase assay showed that ubiquitination-deficient Lck inhibited Lys63-linked polyubiquitination-induced trans-autophosphorylation of Lck by UBR2. Arrow, intact UBR2 protein; asterisk, degraded UBR2 protein.