Fig. 1. The CARD domains of NOD2 interact with the intracellular stalk of CD147. (A) HEK293 cells stably transfected with NOD2 expression construct (HEK^NOD2) or empty vector (HEK^mock) were used for co-immunoprecipitation of CD147 and NOD2. CD147 immunocomplexes and whole cell lysates were assayed for endogenous and precipitated NOD2 and CD147. Precipitates from irrelevant antibody were used as specificity control (ctrl). (B) HEK293 cells were transfected with constructs containing Xpress-tagged NOD2 or NOD2 domains. After precipitation of endogenous CD147, co-precipitated NOD2 or NOD2 domains were visualized using anti-Xpress antibody. Only full-length NOD2 and a fusion protein containing both CARD domains were found in CD147 immmunocomplexes (black arrowheads). (C) HEK293 cells were transfected with constructs containing Xpress-tagged NOD2 or NOD2 domains in combination with a construct containing the intracellular domain of CD147 (IC) fused to EGFP. After precipitation of Xpress-tagged NOD2 domains, EGFP-CD147-IC was identified in the immunocomplexes using anti-GFP antibody. Only full-length NOD2 and both CARD domains were able to precipitate with GFP-CD147-IC (indicated by black arrowheads). Antibody alone (Ab) was used as control. (D) CD147 immunocomplexes derived from THP1 monocytes were used to verify the interaction of CD147 with endogenous NOD2. Detection of two specific bands by use of NOD2 antibody points to the presence of more than one NOD2 isoform.