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Evidence for crossreactivity of JAM‐C antibodies: Implications for cellular localization studies
Author(s) -
Betanzos Abigail,
Liang Tony A.,
Severson Erick A.,
Parkos Charles A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1120.13
Subject(s) - monoclonal antibody , western blot , polyclonal antibodies , microbiology and biotechnology , antibody , immunoprecipitation , transfection , chemistry , immunofluorescence , messenger rna , blot , downregulation and upregulation , cell culture , biology , immunology , biochemistry , gene , genetics
Junctional Adhesion Molecule C (JAM‐C) is implicated in regulation of leukocyte migration, but detailed cellular localization studies are incomplete. Here, we used monoclonal (LUCA14, MAB1189 and Gi11) and polyclonal (AF11889 and 40–9000) antibodies to evaluate JAM‐C expression in some epithelial cell lines. By immunofluorescence (IF), all antibodies showed intercellular junctional staining and western blot (WB) analyses revealed a prominent band at 52kD which is larger than expected for JAM‐C (37kD). Interestingly, RT‐PCR revealed no JAM‐C transcripts in SK‐CO15 (SK), T84, HeLa and HPEF‐II cells, whereas abundant mRNA in platelets, Caco‐2, ARPE and JAM‐C transfected SK (JC‐SK) cells was detected. Using another recently described monoclonal antibody termed PACA4 (Raven biotech), WB of platelets, ARPE and JC‐SK cells revealed a single 37kD band, and it did not label epithelial cells that lack JAM‐C mRNA. Analyses by mass spectrometry identified the 52kD band as cytokeratin 8 (CK8). Further, siRNA‐mediated downregulation of CK8 in JAM‐C mRNA‐negative cells resulted in diminished junctional staining along with a reduction in the 52kD band. These results demonstrate that several anti‐JAM‐C antibodies crossreact with CK8 and suggest that cellular localization studies with such reagents should be interpreted with caution. Our results also suggest that mAb PACA4 is monospecific for JAM‐C.