Cytokeratin 12 in human ocular surface epithelia is the antigenreactive with a commercial anti-Gαq antibody

[摘要] Purpose: In our initial attempt to identify differentiation markersfor ocular surface epithelia, we observed a unique staining pattern by acommercial anti-Gαq antibody. We further isolate and characterizethe protein reactive with this anti-Gαq antibody in human ocularsurface epithelia.Methods: Human donor corneoscleral buttons were sectioned andstained with a battery of commercial antibodies against Gαproteins. Western blot analysis of cell lysates of corneal epithelialcells and HEK 293 cells transfected with Gαq cDNA was used todetermine the identity of the protein reactive with the anti-Gαqantibody (E-17). Comparisons were made with another anti-Gαqantibody (G4415) and an anti-cytokeratin 12C (J7) antibody. The isolatedproteins reactive with E17 and J7 were then analyzed with twodimensional isoelectric focusing. Polypeptide sequences were identifiedusing matrix assisted laser desorption/ionization time of flight massspectrometry (MALDI-TOF MS) after in-gel protein digestion.Results: The E-17 anti-Gαq antibody preferentially stained theentire corneal epithelia and the suprabasal layers of the limbus withcomplete absence of staining in the basal limbus and conjunctiva.Western blot analysis of corneal epithelial cells showed that E-17antibody identified a protein with a molecular weight of 55 kDa.However, the antibody did not react with the purported antigen, Gαq protein (42 kDa) produced by Gαq cDNA. Another anti-Gαqantibody (G4415) did not react with the 55 kDa protein but did reactwith the 42 kDa Gαq protein. Further comparison of the E-17antibody with the J7 antibody revealed that both recognized the 55 kDaband in one and two dimensional analysis. MALDI-TOF MS analysisconfirmed that the 55 kDa protein of interest was actually cytokeratin12 (CK12), rather than Gαq protein.Conclusions: The commercial E-17 anti-Gαq antibody did notreact with Gαq protein, its purported antigen. Instead, itrecognized a 55 kDa protein, which was characterized to be cytokeratin12 by isoelectric focusing and peptide fingerprinting with massspectrometry. Based on its reactivity with CK12, this commercial E-17can be used as a differentiation marker to study ocular surfaceepithelia.

[学科分类] 生物化学/生物物理