Potential misidentification of cyclooxygenase-2 by Western blot analysis and prevention through the inclusion of appropriate controls.
Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.
Liu
Yen-Ting
YT
Department of Molecular Microbiology and Immunology, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90089-9094, USA.
Kardosh
Adel
A
Cooc
Janine
J
Schönthal
Axel H
AH
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D045744:Cell Line, Tumor / Q000201:enzymology; D003429:Cross Reactions; D051546:Cyclooxygenase 2 / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016373:DNA, Antisense / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005189:False Positive Reactions; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012107:Research Design; D013045:Species Specificity; D014162:Transfection
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000911:Antibodies, Monoclonal; D000941:Antigens; D016373:DNA, Antisense; D018076:DNA, Complementary; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human