Protocol for monitoring the stability of transcription factor EB using global protein stability assay

  • STAR Protoc. 2025 Oct 11;6(4):104141. doi: 10.1016/j.xpro.2025.104141.
Boju Wang  1 ,  Yueling Zhao  1 ,  Ruiyan Li  1 ,  Xinying Li  1 ,  Cheng Dong  1 ,  Wenyi Mi  2 ,  Xiaolu Wang  3
Affiliations
  • 1. Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), State Key Laboratory of Experimental Hematology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
  • 2. Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), State Key Laboratory of Experimental Hematology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China. Electronic address: [email protected].
  • 3. Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Tianjin Medical University, Tianjin 300070, China. Electronic address: [email protected].
PMID: 41076632 DOI: 10.1016/j.xpro.2025.104141
Abstract

Transcription factor EB (TFEB), the master regulator of autophagy-lysosomal networks, undergoes multiple regulatory mechanisms to maintain proteostasis. Here, we present a protocol for quantifying TFEB stability following loss of function of its regulator (YEATS domain-containing protein 2 [YEATS2] knockdown), utilizing a dual-fluorescent reporter system based on flow cytometry. We describe strategies for Vector Construction, Cell Transfection, and fluorescence detection. We then detail procedures for analytical workflows. This protocol is designed to enhance sensitivity and reduce processing duration. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Keywords
Cell-based Assays; Flow Cytometry; High Throughput Screening.
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