Work

Protocol to investigate the biochemical details of immune checkpoint ligand/receptor ubiquitination using in vitro ubiquitination assay

Year: 2025

Type: article

Abstract: The activity and stability of immune checkpoint ligands/receptors, including PD-L1 and PD-1, are tightly regulated by ubiquitination. Here, we present a protocol for detecting ubiquitination of the cytoplasmic domain of PD-L1 by various E3 ligases and evaluating the effects of phosphorylation and membrane association on PD-L1 ubiquitination. We describe steps for expressing and purifying recombinant cytoplasmic domain of PD-L1 and related ubiquitination enzymes, preparing liposomes from DC2.4 cells, and detecting PD-L1 ubiquitination using in vitro ubiquitination assays. For complete details on the use and execution of this protocol, please refer to Xie et al. 1 • Instructions for purifying the cytoplasmic domain of immune checkpoints • Purification of CUL3/RBX1 complex from insect cells for in vitro neddylation assay • Steps for the isolation of liposomes from DC2.4 cells • Procedures to detect PD-L1 polyubiquitination by different E3 ligases in vitro Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The activity and stability of immune checkpoint ligands/receptors, including PD-L1 and PD-1, are tightly regulated by ubiquitination. Here, we present a protocol for detecting ubiquitination of the cytoplasmic domain of PD-L1 by various E3 ligases and evaluating the effects of phosphorylation and membrane association on PD-L1 ubiquitination. We describe steps for expressing and purifying recombinant cytoplasmic domain of PD-L1 and related ubiquitination enzymes, preparing liposomes from DC2.4 cells, and detecting PD-L1 ubiquitination using in vitro ubiquitination assays.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104150