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Methods of Assessing STING Activation and Trafficking

  • Vladislav Pokatayev
  • Nan YanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1656)

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.

Key words

Sting Interferon response Innate immunity Cytosolic DNA sensing 

Notes

Acknowledgment

This work is supported by US National Institute of Health (AI098569, AR067135 to N.Y.), UT Southwestern Immunology graduate program training grant (2T32AI005284 to V.P.), and Burroughs Wellcome Fund (N.Y.). The authors have no conflict of interest.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of Texas Southwestern Medical CenterDallasUSA

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