Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells

doi:10.1083/jcb.111.1.95

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Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells

J Vandekerckhove, G Bauw, K Vancompernolle, B Honore and J Celis
Laboratory of Genetics, State University Gent, Belgium.

A systematic comparison of the protein synthesis patterns of cultured normal and transformed human fibroblasts and epithelial cells, using two-dimensional gel protein analysis combined with computerized imaging and data acquisition, identified a 90-kD protein (SSP 5714) as one of the most striking downregulated markers typical of the transformed state. Using the information stored in the comprehensive human cellular protein database, we found this protein strongly expressed in several fetal tissues and one of them, epidermis, served as a source for preparative two-dimensional gel electrophoresis. Partial amino acid sequences were generated from peptides obtained by in situ digestion of the electroblotted protein. These sequences identified the marker protein as gelsolin, a finding that was confirmed by two-dimensional immunoblotting of human MRC-5 fibroblast proteins using specific antibodies and by coelectrophoresis with purified human gelsolin. These results suggest that an important regulatory protein of the microfilament system may play a role in defining the phenotype of transformed human fibroblast and epithelial cells in culture.
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