STING/TMEM173 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

STING (stimulator of interferon genes) is encoded by the TMEM173 gene and is an adaptor molecule involved in the activation of innate immune responses to PAMPS (pathogen-associated molecular patterns) and DAMPS (damage-associated molecular patterns). STING specifically recognizes cytosolic DNA products derived from pathogens (e.g., cytomegalovirus, vaccinia virus, Listeria monocytogenes) or dead cells (1, 2). In the STING pathway, dsDNA derived from pathogens or damaged cells serves as a substrate for the enzyme cGAS (cyclic GMP-AMP synthase) which produces the second messenger cyclic GMP-AMP (cGAMP) from ATP and GTP (3, 4). Under steady-state conditions STING (theoretical molecular weight 42 kDa), a protein localizes to the ER membrane. Upon activation by dsDNA derived second messenger (cGAMP), STING translocates to the Golgi apparatus as a homodimer. Once STING has trafficked to the perinuclear region, it activates TANK binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3) and NF-kB leading to the production of cytokines (e.g., type I interferon) (2, 4). Mutations in the TMEM173 gene affecting STING expression are associated with the development of the auto-inflammatory disease SAVI (STING-associated vasculopathy with onset in infancy) (2). A novel SAVI dominant mutation in the TMEM173 human gene (V155M) leads to increased localization of STING to the Golgi and perinuclear region, indicative of an activated state (1). Hallmarks of SAVI, a rare inflammatory disease, include severe vasculitis in extremities and lung inflammation (7).

References

1. Patel, S., & Jin, L. (2019). TMEM173 variants and potential importance to human biology and disease. Genes and Immunity. https://doi.org/10.1038/s41435-018-0029-9

2. Jounai, N., Kobiyama, K., Takeshita, F., & Ishii, K. J. (2013). Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2012.00168

3. Xiao, T. S., & Fitzgerald, K. A. (2013). The cGAS-STING Pathway for DNA Sensing. Molecular Cell. https://doi.org/10.1016/j.molcel.2013.07.004

4. Kato, K., Omura, H., Ishitani, R., & Nureki, O. (2017). Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA. Annual Review of Biochemistry. https://doi.org/10.1146/annurev-biochem-061516-044813

5. Crowl, J. T., Gray, E. E., Pestal, K., Volkman, H. E., & Stetson, D. B. (2017). Intracellular Nucleic Acid Detection in Autoimmunity. Annual Review of Immunology. https://doi.org/10.1146/annurev-immunol-051116-052331

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-17038

• STING/TMEM173 Antibodies
• STING/TMEM173 Lysate
• STING/TMEM173 Proteins