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Methods in molecular biology (Clifton, N.J.)

Cell-based reporter assay to analyze activation of Nod1 and Nod2.


PMID 21701969

Abstract

Nod1 and Nod2 are pattern recognition receptors of the mammalian innate immune system. They respond to bacterial peptidoglycan fragments and are implicated in host defense against a variety of -different bacterial pathogens. Recent studies furthermore support additional functions of these proteins in the control of adaptive immune responses and intestinal homeostasis. Activation of Nod1 and Nod2 by their cognate elicitors triggers inflammatory responses driven by the activation of NF-κB and MAPK pathways. In this chapter, we describe a quick and reliable cell-based assay using a luciferase reporter to measure Nod1- and Nod2-mediated NF-κB activation. The described protocol was successfully applied to analyze the influences of overexpressed proteins and siRNA-mediated knock-down to provide new insights into the regulation of Nod1/2-specific signaling pathways. Furthermore, this method is well suited for downscaling to high-throughput screening applications.