Proximity Ligation Assay (PLA) to Determine the Endosomal Localization of ESCRT Subunit in Virus-Infected Cells

Methods Mol Biol. 2019:1998:63-72. doi: 10.1007/978-1-4939-9492-2_5.

Abstract

Proximity ligation assay (PLA) is a newly developed technique that outperforms the traditional immunoassays for visualizing the in situ endogenous protein-protein interactions and localizations and the activation of proteins in cell culture systems as well as in tissue sections. PLA, when combined with cellular marker staining, becomes a powerful approach to identify differential interaction of the proteins of endosomal sorting complex required for transport (ESCRT) at distinct stages of virus infection. In this chapter, we describe a PLA protocol to study the localization and interaction between the ESCRT protein TSG101 and endosomal markers in early stages of viral endocytosis in in vitro infected cells.

Keywords: Colocalization; ESCRT; Endocytosis; Proximity ligation assay; Trafficking; Virus infection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Cell Culture Techniques / methods
  • Cell Line
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism*
  • Endosomal Sorting Complexes Required for Transport / immunology
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Endosomes / metabolism*
  • Endothelial Cells / virology
  • Herpesvirus 8, Human / immunology*
  • Humans
  • Protein Interaction Mapping / methods*
  • Staining and Labeling / methods
  • Transcription Factors / immunology
  • Transcription Factors / metabolism*
  • Virus Internalization

Substances

  • DNA-Binding Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Transcription Factors
  • Tsg101 protein