Open AccessMethod for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages
Author(s) -
Gaurav Kumar,
Subhash B. Arya,
Amit Tuli
Publication year - 2018
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.3123
Subject(s) - microbiology and biotechnology , tlr4 , tlr9 , signal transduction , biology , macrophage , mapk/erk pathway , pathogen associated molecular pattern , innate immune system , lipopolysaccharide , toll like receptor , pattern recognition receptor , kinase , receptor , immunology , gene , gene expression , biochemistry , dna methylation , in vitro
Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like Salmonella typhimurium and Escherichia coli . LPS is recognized by TLR4 present on the plasma membrane of macrophages, and LPS-TLR4 association leads to activation of signaling cascades including MAPK phosphorylation, which in turn promotes macrophage activation and microbial killing. This protocol describes the method for studying the role of a gene of interest in Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling, induced by bacterial infection in primary bone-marrow derived macrophages (BMDMs).