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ÀúÀÚ: MK Ki1, MH Jeoung1, JR Choi1, S-S Rho2, Y-G Kwon2, H Shim3, J Chung4, HJ Hong5, BD Song1 and S Lee1 Àú³Î: Oncogene (2013), 1–9 »ç¿ëÇÑ Á¦Ç°: Dynabeads M-270 Epoxy (Cat# 14301, 14302D)
Abstract
It has been suggested that clec14a may be involved in tumor angiogenesis. However, a molecular mechanism has not been clearly identified. In this study, we show for the first time that C-type lectin-like domain (CTLD) of clec14a may be important for regulating cell migration and filopodia formation. Using phage display technology, recombinant human antibodies specific to the CTLDs of human and mouse clec14a (clec14a-CTLD (immunoglobulin G) IgG) were selected. Functional assays using the antibodies showed that clec14a-CTLD IgGs specifically blocked endothelial cell migration and tube formation without affecting cell viability or activation. Further, clec14a-CTLD IgGs inhibited clec14a-mediated cell–cell contact by blocking interaction between CTLDs. Finally, clec14a cross-linking by the clec14a-CTLD IgGs significantly downregulated clec14a expression on the surface of endothelial cells. These results strongly suggest that the clec14a-CTLD may be a key domain in angiogenesis, and that clec14a-CTLD IgGs specifically inhibit angiogenesis by modulating CTLD-mediated cell interactions and clec14a expression on the surface of endothelial cells.
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Phage display Three rounds of biopanning were carried out with immunotubes (Immunotube maxisorp, Nunc, Rochester, NY, USA) or magnetic beads (Dynabeads M-270 epoxy, Invitrogen) coated with 4 mg recombinant hCTLD-Fc or mCTLD-Fc to select clones with cross-species reactivity, as described previously. Ninety-six phage clones were randomly selected from colonies grown on output plates and tested for reactivity to human and mouse CTLDs by phage enzyme immunoassay. DNA of the final scFv clones was sequenced and classified as four scFv clones with different complementarity-determining-region sequences. Á¦Ç° Cat#: 14301, 14302D |
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