Protocol for monitoring the endosomal trafficking of membrane proteins in mammalian cells
Year: 2025
Type: article
Abstract: Here, we present a protocol to study epidermal growth factor (EGF) receptor (EGFR) or transferrin trafficking in mammalian cells. We describe steps for using fluorescent ligands or antibodies, confocal imaging, and quantitative analysis to track their movement. We detail procedures for cell culture preparation, labeling membrane proteins, optimizing imaging, and isolating cell lysates for the biochemical analysis of EGFR degradation after EGF treatment. This protocol is adaptable to various cell types and for assessing genetic or pharmacological impacts on endosomal trafficking. For complete details on the use and execution of this protocol, please refer to Ye et al., 1 Ye et al., 2 and Wang et al. 3 • Instructions for labeling EGFR or transferrin receptor on the cell surface of live cells • Steps for assessing the endosomal trafficking of membrane proteins • Guidance on assessing the degradation of EGFR after EGF treatment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to study epidermal growth factor (EGF) receptor (EGFR) or transferrin trafficking in mammalian cells. We describe steps for using fluorescent ligands or antibodies, confocal imaging, and quantitative analysis to track their movement. We detail procedures for cell culture preparation, labeling membrane proteins, optimizing imaging, and isolating cell lysates for the biochemical analysis of EGFR degradation after EGF treatment. This protocol is adaptable to various cell types and for assessing genetic or pharmacological impacts on endosomal trafficking.
Source: STAR Protocols
Authors: Peng Xu, Jianbo Yue
Institution: Duke Kunshan University
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Awards: G1052826632, G1242311064, Autologous-Allogeneic Human Peripheral CAK-NK Cells Construction and Combination with Immune Checkpoint Inhibitors on Treatment of Genitourinary Tumors: Key Technology Innovation, Efficacy and Mechanistic Study, Development of Bisbenzylisoquinoline Alkaloid-Berbamine and Its Analogs to Prevent and Treat COVID-19 by Compromising the Endosome-lysosome Transport of ACE2, Development Of A Novel Therapeutic To Promote Anticancer Immunity By Manipulating Endosomal Trafficking Of PD-L1, G5642205503
OpenAlex ID: W4408256033