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. Biochemical analysis of their ubiquitination using in vitro enzymatic assays represents an indispensable technique for validating E3 ligase activity and elucidating the molecular mechanisms. However, such analysis has frequently been omitted. This protocol details a method for detecting ubiquitination of the cytoplasmic domain of PD-L1 by various E3s and evaluating the effects of phosphorylation and membrane association on PD-L1 ubiquitination.