BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)

Abstract

Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods. Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods. The presence of pathogenic or self-DNA in the cytosol is a danger signal that triggers the host innate immune system and leads to the production of cyclic GMP-AMP (cGAMP) (1Wu J. Sun L. Chen X. Du F. Shi H. Chen C. Chen Z.J. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA.Science. 2013; 339 (23258412): 826-83010.1126/science.1229963Crossref PubMed Scopus (1344) Google Scholar, 2Sun L. Wu J. Du F. Chen X. Chen Z.J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.Science. 2013; 339 (23258413): 786-79110.1126/science.1232458Crossref PubMed Scopus (2508) Google Scholar, 3Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Röhl I. Hopfner K.P. Ludwig J. Hornung V. cGAS produces a 2‘-5‘-linked cyclic dinucleotide second messenger that activates STING.Nature. 2013; 498 (23722158): 380-38410.1038/nature12306Crossref PubMed Scopus (922) Google Scholar, 4Ablasser A. Chen Z.J. cGAS in action: expanding roles in immunity and inflammation.Science. 2019; 363: eaat865710.1126/science.aat8657Crossref PubMed Scopus (341) Google Scholar). cGAMP functions as a second messenger that binds to and activates the adaptor protein STING, which consists of a four-pass transmembrane domain at the N terminus and a cytosolic cGAMP-binding domain at the C terminus (5Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455 (18724357): 674-67810.1038/nature07317Crossref PubMed Scopus (1945) Google Scholar, 6Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461 (19776740): 788-79210.1038/nature08476Crossref PubMed Scopus (1664) Google Scholar, 7Gao P. Ascano M. Zillinger T. Wang W. Dai P. Serganov A.A. Gaffney B.L. Shuman S. Jones R.A. Deng L. Hartmann G. Barchet W. Tuschl T. Patel D.J. Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA.Cell. 2013; 154 (23910378): 748-76210.1016/j.cell.2013.07.023Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 8Motwan M. Pesiridis S. Fitzgerald K.A. DNA sensing by the cGAS-STING pathway in health and disease.Nat. Rev. Genet. 2019; 20 (31358977): 657-67410.1038/s41576-019-0151-1Crossref PubMed Scopus (456) Google Scholar). Upon binding to cGAMP, inactive dimeric STING undergoes a conformational change, forms oligomers (9Shang G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567 (30842659): 389-39310.1038/s41586-019-0998-5Crossref PubMed Scopus (239) Google Scholar, 10Zhang C. Shang G. Gui X. Zhang X. Bai X.C. Chen Z.J. Structural basis of STING binding with and phosphorylation by TBK1.Nature. 2019; 567 (30842653): 394-39810.1038/s41586-019-1000-2Crossref PubMed Scopus (317) Google Scholar, 11Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178 (31230712): 290-30110.1016/j.cell.2019.05.036Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), and translocates from the endoplasmic reticulum (ER) to the Golgi apparatus (12Saitoh T. Fujita N. Hayashi T. Takahara K. Satoh T. Lee H. Matsunaga K. Kageyama S. Omori H. Noda T. Yamamoto N. Kawai T. Ishii K. Takeuchi O. Yoshimori T. et al.Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response.Proc. Natl. Acad. Sci. U S A. 2009; 106 (19926846): 20842-2084610.1073/pnas.0911267106Crossref PubMed Scopus (573) Google Scholar), where STING is palmitoylated (13Mukai K. Konno H. Akiba T. Uemura T. Waguri S. Kobayashi T. Barber G.N. Arai H. Taguchi T. Activation of STING requires palmitoylation at the Golgi.Nat. Commun. 2016; 7 (27324217): 1193210.1038/ncomms11932Crossref PubMed Scopus (283) Google Scholar) and causes recruitment and activation of the serine/threonine kinase TANK-binding kinase 1 (TBK1) (10Zhang C. Shang G. Gui X. Zhang X. Bai X.C. Chen Z.J. Structural basis of STING binding with and phosphorylation by TBK1.Nature. 2019; 567 (30842653): 394-39810.1038/s41586-019-1000-2Crossref PubMed Scopus (317) Google Scholar). Activated TBK1 then phosphorylates the transcription factor interferon regulatory factor 3 (IRF3) to induce the expression of type I interferon (IFN) and chemokine genes (14Tanaka Y. Chen Z.J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway.Sci. Signal. 2012; 5 (22394562): ra2010.1126/scisignal.2002521Crossref PubMed Scopus (678) Google Scholar, 15Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. Du F. Ren J. Wu Y.T. Grishin N.V. Chen Z.J. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347 (25636800): aaa263010.1126/science.aaa2630Crossref PubMed Scopus (941) Google Scholar). After activation at the Golgi, STING moves to endosomes and then to lysosomes, where STING is ubiquitinated and recruits the autophagy receptor p62/sequestosome 1 (SQSTM1) that leads to the degradation of STING to prevent the excessive production of IFN (16Gonugunta V.K. Sakai T. Pokatayev V. Yang K. Wu J. Dobbs N. Yan N. Trafficking-mediated STING degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response.Cell Rep. 2017; 21 (29241549): 3234-324210.1016/j.celrep.2017.11.061Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 17Prabakaran T. Bodda C. Krapp C. Zhang B-C. Christensen M.H. Sun C. Reinert L. Cai Y. Jensen S.B. Skouboe M.K. Nyengaard J.R. Thompson C.B. Lebbink R.J. Sen G.C. Loo G. et al.Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.EMBO J. 2018; 37: e9785810.15252/embj.201797858Crossref PubMed Scopus (189) Google Scholar, 18Taguchi T. Mukai K. Innate immunity signalling and membrane trafficking.Curr. Opin. Cell Biol. 2019; 59 (30875551): 1-710.1016/j.ceb.2019.02.002Crossref PubMed Scopus (43) Google Scholar). To understand the molecular mechanisms of how the STING pathway is tightly regulated by membrane trafficking, it is important to globally identify proteins that spatiotemporally interact with STING in living cells. BioID is a unique and powerful technique to identify transiently or indirectly interacting proteins in living cells, and it is based on the use of the biotin with M. biotin protein and interacting proteins in Cell Biol. 2012; PubMed Scopus Google Scholar). a protein of to is in cells, it of proteins the of proteins in living Cell Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, the protein by with 2016; PubMed Scopus Google Scholar, to the using biotinylation to protein and Opin. Biol. 2019; PubMed Scopus Google Scholar). The identification of biotinylated proteins by with streptavidin beads by analysis J. Y. Wu Q. L. D. M. M. M. et dynamic protein interaction of the 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, A. D.J. R.J. of an 2016; PubMed Scopus Google Scholar, T. H. T. Y. O. T. T. Structural basis of by into a of the 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). However, biotinylated peptides are rarely detected in conventional method because of the of streptavidin for As a result, nonbiotinylated peptides are identified, and cannot be distinguished from peptides of nonspecifically pull-downed proteins. To an approach using to enrich and identify biotinylated peptides from of proteins K. T. S. O. K. K. D. T. V.K. to biotin of biotinylation on 2017; PubMed Scopus Google Scholar, S. S. Wu X. O. A. a method for and of 2018; PubMed Scopus Google Scholar). of biotinylation on proteins and and proteins and protein However, of biotinylated peptides from the requires which the of nonbiotinylated we the BioID method to efficiently and specifically enrich biotinylated peptides by using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. avidin-like proteins, Tamavidin 1 and Tamavidin were from the Y. M. J. S. Y. N. M. Yamamoto T. avidin-like biotin-binding proteins from the J. 2009; PubMed Scopus Google Scholar), and into Tamavidin to the Tamavidin and biotin Y. K. M. Tamavidin an engineered with reversible biotin-binding 2013; PubMed Scopus Google Scholar). engineered Tamavidin Tamavidin 2-REV, bound to biotinylated which be and specifically by the of using Tamavidin 2-REV biotinylated peptides were identified and quantified from stably expressing STING to T. N. in living and with 2018; PubMed Scopus Google Scholar). the biotinylation of proteins detected in unstimulated cells, STING activation caused the biotinylation of many proteins in the Golgi and endosomes. These proteins included known and of activated The STING in will provide insights into the STING signaling and our approach using Tamavidin 2-REV would to BioID and other identification based on protein biotinylation and and developed which can biotinylation with the BioID T. N. in living and with 2018; PubMed Scopus Google Scholar). To biotin to STING in cells, the BioID to the C terminus or the N terminus of STING or to the N terminus of STING stably in RAW264.7 were with biotin for the biotinylation of many proteins and detected by in expressing a STING P. Ascano M. Zillinger T. Wang W. Dai P. Serganov A.A. Gaffney B.L. Shuman S. Jones R.A. Deng L. Hartmann G. Barchet W. Tuschl T. Patel D.J. Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA.Cell. 2013; 154 (23910378): 748-76210.1016/j.cell.2013.07.023Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, J. S. N. Thompson M. Z. T. Fitzgerald K.A. STING, binds and in to the 2013; PubMed Scopus Google Scholar), to RAW264.7 cells, phosphorylation of TBK1 detected However, phosphorylation of TBK1 by expression of STING or that TurboID-fused STING is were with or for 1 in the presence of biotinylation of many proteins in and unstimulated detected by To protein biotinylation in were with or for 1 in the presence of and with unstimulated cells, and biotinylated proteins a the which is with the of STING at the the other caused the of and biotinylated proteins in a where STING is known to from the its activation These that can and STING-proximal proteins in unstimulated and cells. approach using for and identification of biotinylated peptides developed by K. T. S. O. K. K. D. T. V.K. to biotin of biotinylation on 2017; PubMed Scopus Google Scholar, S. S. Wu X. O. A. a method for and of 2018; PubMed Scopus Google Scholar). that biotinylated peptides be specifically and efficiently by Tamavidin 2-REV with reversible biotin-binding Y. K. M. Tamavidin an engineered with reversible biotin-binding 2013; PubMed Scopus Google Scholar). proteins from or 1 in the presence of biotin in were with and biotinylated peptides were on Tamavidin 2-REV beads the of peptide of Tamavidin 2-REV Y. K. M. Tamavidin an engineered with reversible biotin-binding 2013; PubMed Scopus Google Scholar) is for with peptide The bound biotinylated peptides were with biotin by and we identified and quantified of which were biotinylated peptides biotinylated peptides in the were on analysis of the with identified and quantified of which were biotinylated peptides of biotinylated peptides were identified by with Tamavidin 2-REV Tamavidin 2-REV identification of biotinylated peptides from BioID the Tamavidin biotinylated peptides and As biotinylation of TBK1 by biotinylation of by analysis that biotinylation of proteins by the other biotinylation of and proteins and proteins in by These STING profiles are with the STING trafficking, in which activated STING is from the the Golgi apparatus to endosomes (12Saitoh T. Fujita N. Hayashi T. Takahara K. Satoh T. Lee H. Matsunaga K. Kageyama S. Omori H. Noda T. Yamamoto N. Kawai T. Ishii K. Takeuchi O. Yoshimori T. et al.Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response.Proc. Natl. Acad. Sci. U S A. 2009; 106 (19926846): 20842-2084610.1073/pnas.0911267106Crossref PubMed Scopus (573) Google Scholar, 18Taguchi T. Mukai K. Innate immunity signalling and membrane trafficking.Curr. Opin. Cell Biol. 2019; 59 (30875551): 1-710.1016/j.ceb.2019.02.002Crossref PubMed Scopus (43) Google Scholar). biotinylation of several palmitoyl M. Y. H. R.A. of Full Text Full Text PDF PubMed Scopus Google Scholar) by These proteins are known to be in the Golgi O. F. H. M. Z. F. M. A.A. P. D. D. F. membrane for in the 2018; Full Text Full Text PDF PubMed Scopus Google Scholar) and be in the palmitoylation of STING at the Golgi (13Mukai K. Konno H. Akiba T. Uemura T. Waguri S. Kobayashi T. Barber G.N. Arai H. Taguchi T. Activation of STING requires palmitoylation at the Golgi.Nat. Commun. 2016; 7 (27324217): 1193210.1038/ncomms11932Crossref PubMed Scopus (283) Google Scholar). As in with the palmitoylation phosphorylation of TBK1 and IRF3 in RAW264.7 cells, as in other (13Mukai K. Konno H. Akiba T. Uemura T. Waguri S. Kobayashi T. Barber G.N. Arai H. Taguchi T. Activation of STING requires palmitoylation at the Golgi.Nat. Commun. 2016; 7 (27324217): 1193210.1038/ncomms11932Crossref PubMed Scopus (283) Google Scholar). the of palmitoylation in STING signaling in RAW264.7 STING is known to interact with IRF3 to the phosphorylation of IRF3 (14Tanaka Y. Chen Z.J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway.Sci. Signal. 2012; 5 (22394562): ra2010.1126/scisignal.2002521Crossref PubMed Scopus (678) Google Scholar, 15Liu S. Cai X. Wu J. Cong Q. Chen X. Li T. Du F. Ren J. Wu Y.T. Grishin N.V. Chen Z.J. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation.Science. 2015; 347 (25636800): aaa263010.1126/science.aaa2630Crossref PubMed Scopus (941) Google Scholar), biotinylation of IRF3 detected in and several biotinylated peptides of were by To STING activation causes phosphorylation of RAW264.7 were with in the presence or of the TBK1 and were to K. T. a to 5 Full Text Full Text PDF PubMed Scopus Google Scholar), by with As in an of by of the with protein that the is caused by Furthermore, the of by with that is by the the identified STING-proximal proteins, we on because of its important in antiviral Y. L. D.J. R.J. M. The proteins to and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Chen by cytosolic 7 PubMed Scopus Google Scholar, S. Lee Shi M. M. The antiviral intracellular to 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, G. P. Z. S. The Rep. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). biotinylation of at in the and in the and as a transmembrane protein with N and C a with a cytosolic N terminus and a C terminus G. M. the of antiviral Rev. 1 PubMed Scopus Google Scholar, S. Zhang C. Wang W. Cai X. L. Wu F. Zhang L. C. of and only transmembrane in the Rep. 2016; PubMed Scopus Google Scholar). to the cytosol biotinylation of in the the To the STING and we RAW264.7 stably expressing STING and of with to an T. Chen by cytosolic 7 PubMed Scopus Google Scholar) of STING activation and with at the Upon in the be that the of STING T. Bodda C. Krapp C. Zhang B-C. Christensen M.H. Sun C. Reinert L. Cai Y. Jensen S.B. Skouboe M.K. Nyengaard J.R. Thompson C.B. Lebbink R.J. Sen G.C. Loo G. et al.Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.EMBO J. 2018; 37: e9785810.15252/embj.201797858Crossref PubMed Scopus (189) Google Scholar, 18Taguchi T. Mukai K. Innate immunity signalling and membrane trafficking.Curr. Opin. Cell Biol. 2019; 59 (30875551): 1-710.1016/j.ceb.2019.02.002Crossref PubMed Scopus (43) Google Scholar), where detected To the interaction STING and from were with by with The interaction STING and from 1 to C and phosphorylation of TBK1 at 1 and phosphorylation by with endogenous STING from of RAW264.7 The of at endogenous STING protein at These as a STING at the late activation stage. we developed a simple and method for and identification of biotinylated peptides using Tamavidin To other or proteins that were to biotin Y. K. M. Tamavidin an engineered with reversible biotin-binding 2013; PubMed Scopus Google Scholar), such as M. of biotin-binding by of in J. PubMed Scopus Google Scholar), S. an important in the and of the Sci. PubMed Scopus Google Scholar), and streptavidin with reversible biotin binding Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). However, Tamavidin 2-REV several other proteins for and identification of biotinylated of Tamavidin 2-REV is for with peptides such as peptide the of Tamavidin 2-REV is to Tamavidin 2-REV can be as a protein in and to of biotinylated peptides from peptide identification of biotinylation with of biotinylated proteins K. T. S. O. K. K. D. T. V.K. to biotin of biotinylation on 2017; PubMed Scopus Google Scholar, S. S. Wu X. O. A. a method for and of 2018; PubMed Scopus Google Scholar). Tamavidin of biotinylated peptides is a and method with of biotinylated peptides from requires biotinylated peptides can be specifically from Tamavidin 2-REV by biotin using Tamavidin 2-REV that using which of of biotinylated peptides using Tamavidin 2-REV by identification of biotinylation by would be useful for a of that biotinylation of proteins. These and M.H. V.K. of for and 2015; PubMed Scopus Google biotinylated for Full Text Full Text PDF PubMed Scopus Google Scholar), and of such as palmitoylation J. and PubMed Scopus Google Scholar), and S. S. Wu X. O. A. a method for and of 2018; PubMed Scopus Google Scholar). is known that IRF3 is the transcription factor of the the biotinylation of IRF3 detected is it be because of expression of protein in RAW264.7 other of IRF3 is by TBK1 to induce its translocation and of a to that of of is by TBK1 is by M. D.J. M. N. P. kinase phosphorylation of at induces its and translocation in Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar, J. Chen X. Chen Z.J. is a kinase that Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). mechanisms and functions of the phosphorylation of to be in the the BioID using TurboID-fused STING, we identified the antiviral protein as a STING to functions with and Y. L. D.J. R.J. M. The proteins to and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, G. P. Z. S. The Rep. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, G. M. the of antiviral Rev. 1 PubMed Scopus Google Scholar), we RAW264.7 and However, the and of phosphorylation of TBK1 and degradation of STING the of the interaction STING binding to with phosphorylation of TBK1 at the late activation C and that TBK1 and roles in STING is an antiviral factor and Y. L. D.J. R.J. M. The proteins to and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Chen by cytosolic 7 PubMed Scopus Google Scholar, S. Lee Shi M. M. The antiviral intracellular to 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, G. P. Z. S. The Rep. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). STING is known to be for IFN DNA that STING the of such mechanisms S. S. Patel J.R. T. D. K. D. V. J.R. Barber G.N. A. type I IFN production in by 2012; PubMed Scopus Google Scholar, F. Wang P. Yang L. Yang G. F. W. A. is for of type I interferon and the host antiviral 2013; PubMed Scopus Google Scholar, Z. Jensen Sun C. M.K. A. K. Y. et a STING pathway that controls Commun. 2016; 7 PubMed Scopus Google Scholar, Natl. Acad. Sci. U S A. 2018; PubMed Scopus Google Scholar). STING the antiviral of to the of the on antiviral immune Tamavidin 2-REV beads and were from streptavidin and streptavidin were from and were from and were from The and were from Cell and were from from from RAW264.7 were in RAW264.7 and the stably expressing STING were K. H. Activation of of interferon genes (STING) induces of the immune Biol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). To expressing protein, by to the N terminus of BioID to the N terminus or C terminus of STING, the These were stably into the RAW264.7 using the H. N. N. Y. M. S. M. A. of factor using a to a of PubMed Scopus Google Scholar) by After the of the in To expressing protein, by to the N terminus of STING and into the After by using the system a by To cells, the expressing and targeting for and and expression were into RAW264.7 by using the After of at the expressing were by an Cell and to and the were identified by and DNA were in and and at for were in 1 and with a a and After on for the were at for at The were with or at for 3 After with the the beads were in and at for and were by on a or and a membrane After the membrane with in at by with on the membrane were detected by a of the membrane with or were in and by protein using a After in using a and as H. for protein phosphorylation in 2009; Scholar). with protein were and into using The were with in the presence of 1 for at were at and are were on in a After with or the were with for at and with for at The were with in for 1 at After with the were with and for 1 at in the After with and with the were on the with The were by an were at and are The RAW264.7 expressing were in and The were with or and biotin of to the After 1 of the were with and in and The were by and and then at for at The were and proteins were by and by in After and the proteins were with at The peptide were with and and in for using Tamavidin 2-REV or peptides from the BioID were on of Tamavidin 2-REV beads in the presence of 1 3 of at After with the biotinylated peptides were with of 1 biotin in for at The were using in a and in and peptides from the BioID were on 20 of the beads Cell in the presence of 1 3 of at After with and with the biotinylated peptides were with of in for at The were in and using The were and in and analysis of the peptides on an to a a The peptides were on a with a for by an to for The in with a were with a of an of 1 and a from to were at a of an of 5 an of a of and a of to were the to using with the The were the as an with to of of of as a and of protein N of and biotinylation of as were at a of using the of based on of using the such that the of for peptides the The were by a and the were using the method for the Y. Y. the a and powerful approach to Scholar). for the and of biotinylated The of proteins of biotinylated peptides and STING activation in the BioID of of were using of are The in the via the with the and S. Y. Y. S. Yamamoto T. M. T. Kobayashi D. N. T. N. S. Y. an for 2017; PubMed Scopus Google Scholar). for the and Taguchi for and for and for with of interferon genes endoplasmic reticulum biotin identification TANK-binding kinase 1 1 interferon-induced transmembrane protein 3 cyclic GMP-AMP interferon regulatory factor 3 interferon engineered

Topics & Concepts