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Introduction
Vasohibin-2 (VASH2) belongs to the Vasohibin family which is composed of Vasohibin-1 (VASH1) and VASH2. The human VASH2 gene, firstly identified by Shibuya et al, is reported to be located on chromosome lq32.3 and composed of 355 amino acid residues (1–3). VASH2 is one novel gene homologous to VASH1 and the overall homology between them is >50% at the amino acid level (1,3–5). VEGF is found to induce VASH1 expression in human umbilical vein endothelial cells (HUVECs) (6) similarly to fibroblast growth factor 2 (FGF-2) (7,8). VASH1 was identified to be an intrinsic and specific feedback inhibitor playing an important role in activating ECs engaged in angiogenesis (7,9,10), while exogenous VASH1 hindered sprouting angiogenesis by solid tumors (11,12). In recent studies, it was found that, expression of VASH1 is wide-spread rather than confined to the ECs (13,14). However, unlike VASH1, VASH2 has been found to promote angiogenesis and is identified as an extrinsic and VEGF-independent angiogenic factor (1,15). In recent years, there is increasing research on the interrelation between VASH2 and carcinoma. VASH2 is highly expressed in carcinoma, and functions as a tumor growth promoter by angiogenesis (15,16). The accurate mechanism needs to be further investigated. Currently, there are few VASH2 antibodies in the market which can be applied only in western blotting. The exact intracellular localization of VASH2 is also still unknown, which makes further research on VASH2 difficult.
In our study, we selected New Zealand rabbit as the animal for polyclonal antibody production because it has larger antibody repertoire possessing a higher specificity in recognizing conformational and modified apitopes than do mouse antibody (17–19). We immunized the rabbits separately with a mixture of two specific polypeptides coupled with keyhole limpet hemocyanin (KLH) and a custom-made prokaryotic recombinant part-length VASH2 protein. Antibodies purified from the antiserum were identified by western blotting, immunofluorescence (IF), immunohistochemisty (IHC) and immunoprecipitate (IP). In order to further investigate specific recognition capability of the antibodies, the VASH2 cDNA (encoding for 355 and 311 amino acid residues) was cloned into the Lv-CMV-EGFP vector, respectively. We also synthesized a eukaryotic recombinant VASH2 protein (311 amino acid residues). We found that, VASH2 proteins (355 and 311 amino acid residues) were successfully recognized by our prepared antibodies. IF, IHC and WB (post cytoplasmic/nuclear...