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Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies

Abigail Betanzos1, Michael Schnoor, Eric A Severson

  • 1Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.

Biology of the Cell
|January 16, 2009
PubMed

Insights

Many anti-JAM-C antibodies cross-react with phosphorylated keratin 8, leading to inaccurate cell localization. Only mAb PACA4 is specific for JAM-C, revealing variable expression in epithelial cells at tight junctions.

Area of Science:

  • Cell Biology
  • Immunology
  • Molecular Biology

Background:

  • Junctional adhesion molecule C (JAM-C) is involved in leukocyte migration, cell polarity, spermatogenesis, angiogenesis, and nerve conduction.
  • JAM-C is reportedly found at tight junctions (TJs) and desmosomes, but detailed localization data is lacking.

Purpose of the Study:

  • To evaluate the specificity and localization of commercially available anti-human JAM-C antibodies.
  • To identify potential cross-reacting proteins and clarify the true localization of JAM-C in epithelial cells.

Main Methods:

  • Utilized monoclonal and polyclonal antibodies to assess JAM-C expression in various epithelial cell lines.
  • Performed RT-PCR, immunofluorescence, Western blotting, mass spectrometry, and siRNA-mediated knockdown.
  • Tested antibody specificity against cells with and without JAM-C mRNA.

Main Results:

  • Most anti-JAM-C antibodies showed cross-reactivity with a 52 kDa protein, identified as phosphorylated keratin 8 (K8).
  • The correct 37 kDa JAM-C protein was only detected in cells expressing JAM-C mRNA.
  • Monoclonal antibody PACA4 demonstrated monospecificity for JAM-C, co-localizing with occludin and ZO-1 at TJs.

Conclusions:

  • A majority of anti-human JAM-C antibodies cross-react with phosphorylated K8, necessitating cautious interpretation of localization studies.
  • Only mAb PACA4 is a reliable tool for detecting human JAM-C.
  • JAM-C expression varies across epithelial cell lines and is localized at TJs.
Abstract

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