Analysis of the Localization of NLRs upon Shigella flexneri Infection Exemplified by NOD1

NOD-like receptors (NLRs) are a family of pattern recognition receptors, able to respond to conserved microbial structures and endogenous danger signals. The NLR NOD1 responds to bacterial peptidoglycan, leading to recruitment of RIPK2, following activation of NFκB and MAPK pathways. In this chapter, we describe a fluorescent light microscopic approach to analyze the subcellular distribution of NOD1 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri. This method is based on exogenously expressed EGFP-tagged NOD1 and describes a protocol to obtain inducible cell lines with functional NOD1 signaling. The described protocol is useful to study NOD1 function, also in living cells, using live cell imaging and can be adopted for the study of other NLR proteins.