Protocol for nuclear export signal characterization of cGAS in mammalian cells

STAR Protoc. 2021 Jul 3;2(3):100649. doi: 10.1016/j.xpro.2021.100649. eCollection 2021 Sep 17.

Abstract

The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and triggers the type I interferon production. We used ISD45 or inactivated Vaccinia Virus (VACV) to stimulate cGAS and monitored cellular localization by immunofluorescence microscopy, Operetta high-content screening, and cytoplasmic/nuclear fractionation. LocNES server was used to predict cGAS nuclear export signal (NES) sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. For complete details on the use and execution of this protocol, please refer to Sun et al. Sun et al. (2021).

Keywords: Cell Biology; Immunology; Molecular Biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Mammals
  • Microscopy, Fluorescence / methods*
  • Molecular Biology / methods*
  • Mutagenesis
  • Nuclear Export Signals / genetics
  • Nuclear Export Signals / physiology*
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism*
  • Vaccinia virus / genetics

Substances

  • Nuclear Export Signals
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Nucleotidyltransferases
  • cGAS protein, human

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