Protocol for detecting ligand-induced ubiquitination of LAG3 in human T cells

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.