Abstract

Vasohibin-2 was recently identified as animportant pro-angiogenesis factor in solid tumor and intracellular localizationof its variants is important for elucidating the downstream mechanism(s) of itseffects. Currently there are no reported antibodies affordable forintracellular localization. The aim of this study was to generate andcharacterize polyclonal antibodies against Vasohibin-2 and to determine theintracellular localization of Vasohibin-2. In this study, two polypeptides weresynthesized and one prokaryotic Vasohibin-2 recombinant protein wascustom-made. New Zealand rabbits were immunized with the polypeptide mixtureand prokaryotic recombinant protein, respectively. The purified antibodies fromthe antiserum were validated by ELISA, western blotting (WB),immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation(IP). In order to determine intracellular localization, the cytoplasmic andnuclear proteins of the human liver cancer cell line HepG2 were isolated forthe detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for311 and 355 amino acid residues, fused with or without a DDK/V5 tag at thec-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruseswere successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 aminoacid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag atthe c-terminus) human liver cancer cell lines were established. Approximately1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injectedsubcutaneously into the flanks of BALB/c nude mice. Xenograft tumors wereharvested for immunohistochemistry. HepG2 cells were transiently transfectedwith the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355amino acid residues with a DDK tag at the c-terminal), followed by anti-DDKimmunofluorescence. The antibodies obtained were able to detect human VASH2successfully as applied in western blotting, IF, IHC and IP. Results from IF,IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quitedifferent intracellular localization of VASH2 protein. The VASH2 (with 355amino acid residues) was located in the cytoplasm while VASH2 (with 311 aminoacid residues) was located in the nucleus. The former was found to be a relativelylow abundance protein. We successfully generated three rabbit anti-humanVasohibin-2 polyclonal antibodies which can be used for western blotting, IF,IP and IHC. These antibodies will provide a convenient tool for further studieson Vasohibin-2. This is the first study to report differences in theintracellular localization of the VASH2 protein and, hence, a new researchdirection on the study of VASH2.