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Biochemical Isolation of the Myddosome from Murine Macrophages

  • Yunhao Tan
  • Jonathan C. KaganEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

Ligand-induced macromolecular protein complex formation has emerged as a common means by which the innate immune system activates signal transduction pathways essential for host defense. Despite their structural divergence, key signaling molecules in diverse innate immune pathways mediate signal transduction by assembling higher-order protein complexes at specific subcellular locations in a stimulus-dependent manner. These protein complexes are collectively known as the supramolecular organizing centers (SMOCs), which link active receptors to a variety of downstream cellular responses. In the Toll-like receptor (TLR) pathway, the signaling adaptor MyD88 is the core of a SMOC called the myddosome, which is composed of the sorting adaptor TIRAP and the IRAK family kinases. Depending on the microbial ligands encountered, the myddosome can be assembled at the plasma membrane or endosomes, thereby leading to NF-ĸB and AP-1 activation, and the subsequent expression of pro-inflammatory cytokines. Herein, we provide a detailed protocol for studying myddosome assembly in murine bone marrow-derived macrophages (BMDMs).

Keywords

TLR SMOCs MyD88 Myddosome Immunoprecipitation 

Notes

Acknowledgments

This work was supported by NIH grants AI093589, AI116550, and P30 DK34854 to J.C.K. J.C.K. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Y.T. is supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (the Merck Fellow).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Gastroenterology, Harvard Medical SchoolBoston Children’s HospitalBostonUSA

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