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

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.