AA98 antigen expression analyses and identification of the AA98 antigen as CD146. (A) Flow cytometry analysis of AA98 antigen expression on HUVECs. (Ai) AA98 antigen is not detectable on primary HUVECs (pHUVEC) immediately after isolation. (Aii) mAb AA98 recognizes its antigen on the stimulated HUVECs (sHUVEC) cultured in conditioned medium from hepatocarcinoma SMMC 7721 cells. (Aiii) An increase in mAb AA98 immunoreactivity is detected on primary HUVECs cultured in the presence of 10% FBS and is decreased after shifting the cells to serum-free medium for at least 12 hours. (B) Immunohistochemical analysis of the AA98 antigen on frozen sections from human tumor and normal tissues. AA98 antigen is readily detectable in tumor neovasculature of hepatocarcinoma (Bii), thyroid tumor (Biv), and brain tumor (Bvi), whereas there was no significant mAb AA98 staining in corresponding normal tissue, such as liver (Bi), thyroid (Biii), and brain (Bv). Original magnification, × 200. (C) Characterization, purification, and identification of the AA98 antigen as CD146. (Ci-ii) Western blot analysis of cell surface—biotinylated proteins of HUVECs immunoprecipitated by mAb AA98, separated under nonreducing or reducing conditions by SDS-PAGE, and detected by either HRP-conjugated streptavidin (Ci) or mAb AA98 followed by HRP-conjugated anti—mouse IgG (Cii). Positions of molecular-weight standard marker proteins are shown at the right margin. (Ciii) Comparison of the first 20 amino acids of AA98 antigen with the amino acid sequence of human CD146. The first 20 amino acid residues of AA98 antigen are identical to the amino acid sequence of positions 24 to 43 of the human CD146 protein (Swissprot accession P43121). (Civ) Flow cytometric analysis shows the binding of mAb AA98 to stably CD146-transfected SBcl-2 cells (CD146-SBcl-2) but not to parental CD146-SBcl-2 cells.