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Updated: Jan 17, 2026
Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Published on: May 10, 2022
Protocol for detecting ligand-induced ubiquitination of LAG3 in human T cells
Anran Dai1, Xinnuo Li2, Haopeng Wang3
1Changping Laboratory, Beijing 102206, China.
Ligand-induced ubiquitination plays a critical role in regulating the immune checkpoint function of Lymphocyte Activation Gene 3 (LAG3). Here, we present a protocol for detecting LAG3 ubiquitination in T cells. We describe the establishment of a Jurkat T cell line stably expressing LAG3, followed by detailed assays to measure ligand engagement-dependent LAG3 ubiquitination using the Raji-Staphylococcal enterotoxin E (SEE) system as an antigen-presenting platform. This protocol provides a robust approach for investigating the molecular mechanisms underlying LAG3-mediated immune regulation through post-translational modifications. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.
Ligand-induced ubiquitination plays a critical role in regulating the immune checkpoint function of Lymphocyte Activation Gene 3 (LAG3). Here, we present a protocol for detecting LAG3 ubiquitination in T cells. We describe the establishment of a Jurkat T cell line stably expressing LAG3, followed by detailed assays to measure ligand engagement-dependent LAG3 ubiquitination using the Raji-Staphylococcal enterotoxin E (SEE) system as an antigen-presenting platform. This protocol provides a robust approach for investigating the molecular mechanisms underlying LAG3-mediated immune regulation through post-translational modifications. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.
