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

  • STAR Protoc. 2025 Oct 17;6(4):104150. doi: 10.1016/j.xpro.2025.104150.
Guojiao Xie  1 ,  Lin Gao  2 ,  Linxia Tian  3 ,  Xinning Li  2 ,  Tiantian Zheng  2 ,  Xian Yu  3 ,  Hanjie Jiang  4 ,  Zan Chen  5
Affiliations
  • 1. Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China. Electronic address: [email protected].
  • 2. Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China.
  • 3. Phase I Clinical Trial Center, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
  • 4. Institute of Medical Physiology, Chinese Institutes for Medical Research (CIMR), Beijing 100069, China; School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China.
  • 5. Basic Medicine Research and Innovation Center for Novel Target and Therapeutic Intervention, Ministry of Education, College of Pharmacy, Chongqing Medical University, Chongqing 400016, China; Phase I Clinical Trial Center, The Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. Electronic address: [email protected].
PMID: 41108680 DOI: 10.1016/j.xpro.2025.104150
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.

Keywords
Cell Membrane; Protein Biochemistry; Protein expression and purification.
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