When coupled to other approaches, such as the yeast two-hybrid system, they become an invaluable tool for studying and understanding protein function. Most of the NPRAP-interacting proteins that were previously reported in the literature were almost exclusively associated with the ability of NPRAP to induce dendrite growth. This has clearly limited research on NPRAP function. However, as NPRAP is an armadillo homolog and a PS1 partner, and as reports of its dynamic nucleocytoplasmic shuttling and role in gene regulation have emerged, it has become clear that NPRAP functions are not restricted to cell adhesion and protrusion elaboration. In an effort to address how a brain-specific protein evolved to exert such distinct, yet elaborate roles and to determine how the multiple roles of NPRAP are triggered and mediated, we identified 14 novel NPRAP-binding partners. To our knowledge, this is the first highthroughput proteomic analysis aimed at assessing the NPRAP interactome. Although we used a monoclonal antibody for the protein enrichment and nonspecific binding to IgGs was ruled out, “false” interactions cannot be fully excluded until individual validation using complimentary methods is performed. However, many of the interactions described herein may be stable, because they survived the incubation and washing steps without the use of crosslinking agents. In addition, when proteins display similarity in sequence homology and molecular function, they have a greater probability of being specific interactors. Correspondingly, interferon regulatory factor 2- binding proteins 1 and 2 and enhanced at puberty 1 are such candidates. Whereas enhanced at puberty 1 is a dual transcriptional regulator in the neuroendocrine system, the other two proteins seem to participate in the interferon pathway as co-repressors in an interferon regulatory factor 2-dependent MLN4924 Metabolic Enzyme/Protease inhibitor manner. Interestingly, we recently reported the involvement of NPRAP in transcriptional modulation, including the activation of interferon-inducible genes and the repression of several other targets. Remarkably, the above transcription factors were not the only proteins related to nucleic acid regulation identified in our study. Werner helicase-interacting protein 1 participates in DNA replication through its association with Werner syndrome ATP-dependent helicase, mutations of which result in genomic instability and premature aging. Additionally, poly- binding protein binds the poly tail of mRNAs to regulate translation initiation, mRNA decay and silencing, whereas serine/arginine repetitive matrix 2 protein is a core member of the catalytic spliceosome that regulates the process by which introns are removed from precursor mRNAs. All of the above-mentioned proteins reinforce a role for NPRAP in controlling gene expression. Interestingly, the only structural protein from the cytoskeleton detected in our analysis was the neurofilament subunit, alphainternexin. This neuronal-specific intermediate filament exhibits axonal and dendritic localization and has also been shown to induce neurite outgrowth in PC12 cells and to mediate neurofilament anchorage to membrane-associated proteins and receptors.