Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies
Article first published online: 3 JAN 2012
2009 Société Française des Microscopies and Société Biologie Cellulaire de France
Biology of the Cell
Volume 101, Issue 8, pages 441–453, August 2009
How to Cite
Betanzos, A., Schnoor, M., Severson, E. A., Liang, T. W. and Parkos, C. A. (2009), Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies. Biology of the Cell, 101: 441–453. doi: 10.1042/BC20080130
- Issue published online: 3 JAN 2012
- Article first published online: 3 JAN 2012
- Received 25 November 2008; Accepted 14 January 2009
- antibody specificity;
- epithelial cell;
- junctional adhesion molecule C (JAM-C);
- tight junction
Background information. JAM-C (junctional adhesion molecule C) has been implicated in the regulation of leukocyte migration, cell polarity, spermatogenesis, angiogenesis and nerve conduction. JAM-C has been also reported to concentrate at TJs (tight junctions) and desmosomes, although detailed localization studies remain incomplete.
Results. Monoclonal (LUCA14, MAB1189, Gi11, and PACA4) and polyclonal (40-9000) antibodies were employed to evaluate JAM-C expression/localization in various epithelial cell lines. However, RT—PCR (reverse transcription—PCR) assays revealed no JAM-C mRNA in SK-CO15, HeLa and HPAF-II cells, whereas abundant mRNA was detected in platelets, Caco-2 and ARPE cells. Interestingly, immunofluorescence localization in all cells revealed strong intercellular junctional staining with all of the above antibodies, except PACA4. Given the positive staining results in cells lacking JAM-C mRNA, immunoblot analyses were performed. Western blots revealed a prominent protein band at 52 kDa in all cells tested with all antibodies except PACA4. However, the correct size of JAM-C (37 kDa) was only detected in cells containing 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 identified 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 band. Using an antibody specific for K8 phosphorylated at Ser73, the 52 kDa protein 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, only mAb PACA4 is monospecific for human JAM-C. Analyses using PACA4 reveal that JAM-C expression is variable in different epithelial cell lines with co-localization at TJs.