Fig. 4

MARCH7 directly targeted NOD1. A After co-IP assay, we detected the top 10 UbiBroswer-predicted substrates of MARCH7 using western blot. Here, we studied the interaction between MARCH7 and NOD1 protein. B The degradation of HA-NOD1 by Flag-MARCH7 verified by western blot, and the ubiquitination of HA-NOD1 detected by co-IP assay. C Schematic representation of the domains of MARCH7. D Co-IP detection of each domain of MARCH7 protein interacting with HA-NOD1. E Western blot of the degradation of HA-NOD1 by Flag-MARCH7 mutant (Flag-MARCH7-Mut). Co-IP detection showed that Flag-MARCH7-Mut could not induce the ubiquitination-mediated degradation of HA-NOD1. All experiments were conducted in triplicate, and each assay was repeated independently three times. F Co-IP and in vitro experiments of ubiquitination determined the effects of MARCH7 on NOD1 ubiquitination by using purified MARCH7 protein and NOD1 protein. All experiments were conducted in triplicate, and each assay was repeated 3 times independently