(B) Using ELISA assay, JAM-C antibodies were tested to specifically recognize the recombinant protein JAM-C/hFc or other human recombinant proteins of the JAM family or structurally comparable proteins. all cells tested with all antibodies except PACA4. However, the correct size of JAM-C (37 kDa) was only detected in cells made up of JAM-C mRNA. Immunofluorescence staining of JAM-C mRNA-expressing Caco-2 cells using mAb PACA4 revealed co-localization with occludin and ZO-1 (zonula occludens 1) at TJs. Analyses by MS recognized the cross-reactive 52 kDa protein band as K8 (keratin 8). Furthermore, siRNA (small interfering RNA)-mediated downregulation of K8 in JAM-C mRNA-negative cells resulted in diminished junctional staining along with a reduction in the intensity of the 52 kDa protein Mouse monoclonal to CK17. Cytokeratin 17 is a member of the cytokeratin subfamily of intermediate filament proteins which are characterized by a remarkable biochemical diversity, represented in human epithelial tissues by at least 20 different polypeptides. The cytokeratin antibodies are not only of assistance in the differential diagnosis of tumors using immunohistochemistry on tissue sections, but are also a useful tool in cytopathology and flow cytometric assays. Keratin 17 is involved in wound healing and cell growth, two processes that require rapid cytoskeletal remodeling band. Using an antibody specific for K8 phosphorylated at Ser73, the 52 kDa protein Phenytoin sodium (Dilantin) was identified as this phosphorylated form of K8. == Conclusions == The results from the present study demonstrate that a majority of available anti-human JAM-C antibodies cross-react with phosphorylated K8 and suggest that cellular localization studies using these reagents should be interpreted with caution. Of the JAM-C antibodies tested, Phenytoin sodium (Dilantin) only mAb PACA4 is usually monospecific for human JAM-C. Analyses using PACA4 reveal that JAM-C expression is usually variable in different epithelial cell lines with co-localization at TJs. Keywords:antibody specificity, epithelial cell, junctional adhesion molecule C (JAM-C), keratin, tight junction == Introduction == JAMs (junctional adhesion molecules) are a novel subset of immunoglobulin superfamily proteins that belong to the larger CTX (cortical thymocyteXenopus) family of proteins (Aurrand-Lions et al., 2001a). JAMs are expressed at intercellular junctions of endothelial and epithelial cells, as well as on the surface of leukocytes, platelets and erythrocytes (Naik et al., 1995;Malergue et al., 1998;Martin-Padura et al., 1998;Williams et al., 1999;Ebnet et al., 2004;Mandell and Parkos, 2005). JAM proteins have been implicated in regulation of cell-cell adhesion, barrier function, leukocyte migration, platelet activation, angiogenesis Phenytoin sodium (Dilantin) and retrovirus binding (Ebnet et al., 2004;Mandell and Parkos, 2005). So far, three members of the JAM family have been explained: JAM-A (Martin-Padura et al., 1998), JAM-B (Cunningham et al., 2000;Palmeri et al., 2000) and JAM-C (Aurrand-Lions et al., 2000;Arrate et al., 2001). However, JAMs are closely related to other divergent proteins such as JAM4 (Hirabayashi et al., 2003), JAML (JAM-like) (Moog-Lutz et al., 2003), CLMP [CAR (coxsackie and adenovirus receptor)-like membrane protein] (Raschperger et al., 2004), CAR (Bergelson et al., 1997) and ESAM (endothelial cell adhesion molecule) (Hirata et al., 2001). Human JAM-C cDNA encodes a 310 residue precursor protein with a molecular mass of 35 kDa, which includes an extracellular domain name consisting of an N-terminal transmission peptide, two Ig-like domains, two potentialN-linked glycosylation sites, a single membrane-spanning region and a short cytoplasmic tail enclosing a PDZ-binding motif and a PKC (protein kinase C) phosphorylation consensus site (Arrate et al., 2001). JAM-C has been shown to interact with PAR (protease-activated receptor)-3 and ZO-1 (zonula occludens 1) in endothelial cells and with PAR-6, Cdc42, PKC, PATJ (Pals1-associated TJ protein), and RA175 in spermatids, where JAM-C functions as scaffold to recruit polarity complexes to TJs (tight junctions) (Ebnet et al., 2003;Gliki et al., 2004;Mirza et al., 2006;Fujita et al., 2007). Furthermore, targeted disruption of JAM-C revealed a crucial role for this protein in spermatogenesis and maintaining the integrity and function of myelinated peripheral nerves, since JAM-C deficient mice are infertile due to an inability to produce mature spermatozoa (Gliki et al., 2004). Additionally, JAM-C knockout mice exhibit loss of myelin sheath integrity and display nerve conduction deficits (Scheiermann et al., 2007). It has been reported that JAM-C is usually widely expressed in endothelial cells, platelets, T-cells and NK (natural killer) cells, and in tissues including intestine, skin, heart, lymph nodes, testis, thymus, lung, kidney, liver, placenta, retina, brain and peripheral nerves (Arrate et al., 2001;Aurrand-Lions et al., 2001a,2005;Liang et al., 2002;Santoso et al., 2002;Ebnet et al., 2003;Gliki et al., 2004;Ludwig et al., 2005;Daniele et al., 2007;Scheiermann et al., 2007). In epithelial cells, JAM-C has been reported to be expressed at TJs of RPE (retinal.