We describe a rapid and very sensitive methi$d for detecting proteins as antigens after their separationiin polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethylpaper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been -repared; it does not crossreact with VP1, as demonstrated by t is method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically Specific proteins have been detected in crude extracts by immunoprecipitation with specific antisera, followed by gel electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4). The proteins are usually tagged with a radioisotope to facilitate detection and to distinguish them from the unlabeled proteins of the antisera. Studies of the tumor (T) antigens coded for by SV40 (1, 2) or polyoma (3, 4) provide recent examples of the use of this method. if the antigen is a member of a complex aggregate of different polypeptides, the entire aggregate will be precipitated and detected. Also, proteolytic degradation of the antigens in a crude extract can sometimes be a problem during the long incubation times necessary for complete immunoprecipitation. To circumvent complications such as these, alternative approaches were developed in which unlabeled proteins were first separated by gel electrophoresis and then detected by incubating the gel with radioactive antibody (5-7) or by transferring the bands of protein from the gel into an overlay of agarose impregnated with unlabeled serum (8). However, because careful washing is required to obtain good ratios of signal to background, these procedures are slow.We now describe a modification of the approach of separating the unlabeled proteins first. The procedure is simple, rapid, and very sensitive, and it gives excellent ratios of signal to background. By using this procedure, we show that SV40 T antigen has periodate-sensitive groups which can be major antigenic determinants and that an antiserum directed against the purified SV40 virion protein VP3 does not crossreact with VP1.MATERIALS AND METHODS Cells and Viruses. The CV-1 line of African monkey kidney cells and SV40 strain VA 45-54 (9) were used. SV40 virions were purified according to Christiansen et al. (10). TRK-54, an SV40-transforme...

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Podoplanin is a small cell-surface mucin-like glycoprotein that plays a crucial role in the development of the alveoli, heart, and lymphatic vascular system. Emerging evidence indicates that it is also involved in the control of mammary stem-cell activity and biogenesis of platelets in the bone marrow, and exerts an important function in the immune response. Podoplanin expression is upregulated in different cell types, including fibroblasts, macrophages, T helper cells, and epithelial cells, during inflammation and cancer, where it plays important roles. Podoplanin is implicated in chronic inflammatory diseases, such as psoriasis, multiple sclerosis, and rheumatoid arthritis, promotes inflammation-driven and cancer-associated thrombosis, and stimulates cancer cell invasion and metastasis through a variety of strategies. To accomplish its biological functions, podoplanin must interact with other proteins located in the same cell or in neighbor cells. The binding of podoplanin to its ligands leads to modulation of signaling pathways that regulate proliferation, contractility, migration, epithelial–mesenchymal transition, and remodeling of the extracellular matrix. In this review, we describe the diverse roles of podoplanin in inflammation and cancer, depict the protein ligands of podoplanin identified so far, and discuss the mechanistic basis for the involvement of podoplanin in all these processes.