Methods of assessing STING activation and trafficking

Vladislav Pokatayev, Nan Yan

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

The signaling adapter protein STING is crucial for the host immune response to cytosolic DNA and cyclic dinucleotides. Under basal conditions, STING resides on the endoplasmic reticulum (ER), but upon activation, it traffics through secretory pathway to cytoplasmic vesicles, where STING activates downstream immune signaling. Classical STING activation and trafficking are triggered by binding of cyclic dinucleotide ligands. STING signaling can also be activated by gain-of-function mutations that lead to constitutive trafficking of STING. These gain-of-function mutations are associated with several human diseases such as STING-associated vasculopathy with onset in infancy (SAVI), systemic lupus erythematosus (SLE), or familial chilblain lupus (FCL). This dynamic activation pathway presents a challenge to study. We describe methods here for measuring ligand-dependent and ligand-independent activation of STING signaling in HEK293T cells. We also describe a retroviral-based reconstitution assay to study STING protein trafficking and activation in immune competent cells such as mouse embryonic fibroblasts (MEF), which avoids the use of plasmid DNA. These methods will expedite research regarding STING trafficking and signaling dynamics in the settings of infection and autoimmune diseases.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages167-174
Number of pages8
Volume1656
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1656
ISSN (Print)1064-3745

Fingerprint

Ligands
Cytoplasmic Vesicles
Mutation
Secretory Pathway
DNA
Protein Transport
Endoplasmic Reticulum
Systemic Lupus Erythematosus
Autoimmune Diseases
Plasmids
Fibroblasts
Infection
Research
Proteins
Chilblain lupus 1

Keywords

  • Cytosolic DNA sensing
  • Innate immunity
  • Interferon response
  • Sting

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Pokatayev, V., & Yan, N. (2017). Methods of assessing STING activation and trafficking. In Methods in Molecular Biology (Vol. 1656, pp. 167-174). (Methods in Molecular Biology; Vol. 1656). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7237-1_10

Methods of assessing STING activation and trafficking. / Pokatayev, Vladislav; Yan, Nan.

Methods in Molecular Biology. Vol. 1656 Humana Press Inc., 2017. p. 167-174 (Methods in Molecular Biology; Vol. 1656).

Research output: Chapter in Book/Report/Conference proceedingChapter

Pokatayev, V & Yan, N 2017, Methods of assessing STING activation and trafficking. in Methods in Molecular Biology. vol. 1656, Methods in Molecular Biology, vol. 1656, Humana Press Inc., pp. 167-174. https://doi.org/10.1007/978-1-4939-7237-1_10
Pokatayev V, Yan N. Methods of assessing STING activation and trafficking. In Methods in Molecular Biology. Vol. 1656. Humana Press Inc. 2017. p. 167-174. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7237-1_10
Pokatayev, Vladislav ; Yan, Nan. / Methods of assessing STING activation and trafficking. Methods in Molecular Biology. Vol. 1656 Humana Press Inc., 2017. pp. 167-174 (Methods in Molecular Biology).
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