ZO-3, a ~130kD protein, contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a SH3 domain, and a guanylate kinase-liked domain. Like ZO-1 and ZO-2, ZO-3 contains a C-terminal acidic domain and a basic region (between the first and second PDZ domains), making it belongs to the MAGUK protein family. By Western Blot analysis, ZO-3 is identified as a ~130kD protein that directly interacts with tight junction proteins ZO-1 and occuldin, but not with ZO-2. However, in one study, ZO-2, ZO-3 and Occludin colocalized with actin aggregates at cell border in MDCK cells. ZO-3 is localized to sites of cell-cell interaction, identical to that of ZO-1 in tight junctions. By Immunoprecipitation analysis, ZO-3 co-immunoprecipitates with ZO-1. Using MDCK cells, ZO-3 protein colocalized with ZO-1 by Immunofluorescence. In vitro binding assays, the N-terminal of ZO-3 showed preferably binding of ZO-1 and actin filaments. Moreover, the binding of ZO-1/ZO-2/ZO-3 was also found to interact with certain Claudin proteins.