Protocol for nuclear export signal characterization of cGAS in mammalian cells
| Autor: | Fei Guo, Yu Huang, Myles McLean, Chen Liang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Inactivated Vaccinia Virus
Science (General) viruses Green Fluorescent Proteins Immunology Vaccinia virus General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound Q1-390 Protocol Animals Humans Nuclear export signal Molecular Biology Cellular localization Mammals Nuclear Export Signals General Immunology and Microbiology General Neuroscience Mutagenesis Cell Biology Type I interferon production Nucleotidyltransferases Cell biology chemistry Microscopy Fluorescence Cytoplasm DNA Function (biology) HeLa Cells |
| Zdroj: | STAR Protocols, Vol 2, Iss 3, Pp 100649-(2021) STAR Protocols |
| ISSN: | 2666-1667 |
| Popis: | Summary 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) Graphical abstract Highlights • LocNES is used to predict cGAS nuclear export signal • Validation of cGAS nuclear export signal • cGAS localization detected by microscopy and cell fractionation The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and synthesizes cGAMP, and triggers the type I interferon production. We used ISD45 or inactivated 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 sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. |
| Databáze: | OpenAIRE |
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