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

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

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: 2025130522@stu.cqmu.edu.cn.
  • 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: zanchen@cqmu.edu.cn.
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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