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Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endotheliat cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation atone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells. [ABSTRACT FROM AUTHOR]

Examines the interaction between monoclonal antibodies and vascular endothelial growth factor-D (VEGF-D) receptors. Localization of tyrosine kinases in vascular and lymphatic endothelial cells; Response of microvascular endothelial cells to VEGF-D; Analysis on the biological functions of VEGF-D.

Studies the effect of two monoclonal antibodies P2 (LyP 2) or P4 (LyP 4), specific for the platelet membrane glycoprotein IIb/IIIa complex, on binding of fibrinogen or fibronectin to thrombin-stimulated platelets. Presence of specific regions on the glycoprotein IIb-IIIa complex which act as binding sites for fibrinogen or fibronectin.