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EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB Pathway.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2017; Vol. 1582, pp. 103-108. - Publication Year :
- 2017
-
Abstract
- The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.
- Subjects :
- ErbB Receptors genetics
ErbB Receptors metabolism
HeLa Cells
Humans
Endosomal Sorting Complexes Required for Transport genetics
Endosomal Sorting Complexes Required for Transport metabolism
Epidermal Growth Factor genetics
Epidermal Growth Factor metabolism
Human T-lymphotropic virus 1 genetics
Human T-lymphotropic virus 1 metabolism
Lysosomes genetics
Lysosomes metabolism
Proteolysis
Viral Proteins genetics
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1582
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 28357665
- Full Text :
- https://doi.org/10.1007/978-1-4939-6872-5_8