Work

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

Year: 2025

Type: article

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 • Steps for generation of the GPS reporter cell line • Instructions for fluorescence-based analysis of TFEB stability via flow cytometry • Guidance on real-time quantification of protein turnover dynamics in live cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104141