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

## TL;DR

This paper describes a protocol to detect ligand-induced ubiquitination of LAG3 in T cells, helping to study immune checkpoint regulation.

## Contribution

A detailed and robust protocol for measuring LAG3 ubiquitination in T cells using a Raji-SEE system is introduced.

## Key findings

- A Jurkat T cell line stably expressing LAG3 was established.
- Ligand engagement-dependent LAG3 ubiquitination was measured using the Raji-SEE system.
- The protocol enables investigation of post-translational modifications in LAG3-mediated immune regulation.

## Abstract

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

•Instructions for the detection of ligand-induced LAG3 ubiquitination•Steps for generating stable LAG3-expressing human Jurkat T cells•Procedures for ligand-receptor activation and ubiquitination studies

Instructions for the detection of ligand-induced LAG3 ubiquitination

Steps for generating stable LAG3-expressing human Jurkat T cells

Procedures for ligand-receptor activation and ubiquitination studies

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.