Enteropathogenic Escherichia coli (EPEC) is a pathogen which utilises a type III secretion system (T3SS) to translocate effector proteins directly into host cells. In recent years, several of these effectors have been shown to subvert host cell signaling. Non-LEE encoded effector C (NleC) is one of a number of effectors which inhibit innate immune signaling such as NF-κB signaling. The mechanism of action of NleC is unlike those of effectors such as NleB or NleE which inhibit NF-κB signaling upstream of NF-κB proteins themselves. NleC acts directly on the NF-κB protein p65 by cleaving it, preventing the activation of inflammatory signaling and dampening the immune response to allow EPEC to persist within the host. Analysis of the amino acid sequence of NleC reveals it contains the zinc metalloprotease consensus motif HEXXH. Mutations of the histidines or glutamic acid within this motif abrogate NleC function. Immunoprecipitations using Hek293T cells expressing FLAG-tagged forms of the protease mutant NleCE184A reveal that NleCE184A is able to bind to endogenous p65. Cleavage of p65 has been shown to occur within the Rel homology domain (RHD) of p65 which is highly conserved among all NF-κB proteins. As expected, NleC cleaves other NF-κB proteins such as p50 and c-rel. In this study we show that cleavage of p50 also occurs within the RHD. This study also aims to identify the regions of binding between p65 and NleC. We show that NleC is likely to bind between amino acids 19 and 29 of p65. Current studies are underway to further define this binding and obtain a better understanding of substrate recognition by NleC.