NOD2 is ubiquitinated.

A) To determine NOD1 and NOD2 ubiquitination, denatured lysates of HEK293T cells expressing the indicated proteins were subjected to immunoprecipitation. Immunoprecipitates were immunoblotted using the indicated antibodies B) To determine the type of ubiquitin-linkage, lysates of HEK293T cells expressing the NOD1 or NOD2 were subjected to immunoprecipitation. Endogenous ubiquitin was revealed using a K48-link specific antibody. C) HEK293T cells were transfected for 72 h with siCTR, siTRIM27-1 or -3 after expression of Flag-NOD2 and HA-Ubiquitin. Immunoblots of immunoprecipitates (IP) and total lysates (Input) were performed using the indicated antibodies. D) TRIM27 and a E3 mutant of TRIM27 were expressed together with NOD2 and HA-ubiquitin in HEK293T cells. Immunoprecipitates were probed with the indicated antibodies. E) HEK293T cells expressing the indicated proteins were stimulated with 10 µM MDP for the indicated time. Lysates were subjected to immunoprecipitation as described in (C). Densitometric analysis of the TRIM27 signal in the immunoprecipitation normalized to the input signal is shown (bottom). Representative data of at least three independent experiments are shown.