Generation and characterization of rabbit polyclonal antibodies against Vasohibin-2 for determination of its intracellular localization

Abstract

Vasohibin-2 was recently identified as an important pro-angiogenesis factor in solid tumor and intracellular localization of its variants is important for elucidating the downstream mechanism(s) of its effects. Currently there are no reported antibodies affordable for intracellular localization. The aim of this study was to generate and characterize polyclonal antibodies against Vasohibin-2 and to determine the intracellular localization of Vasohibin-2. In this study, two polypeptides were synthesized and one prokaryotic Vasohibin-2 recombinant protein was custom-made. New Zealand rabbits were immunized with the polypeptide mixture and prokaryotic recombinant protein, respectively. The purified antibodies from the antiserum were validated by ELISA, western blotting (WB), immunofluorescence (IF), immunohistochemistry (IHC) and immunoprecipitation (IP). In order to determine intracellular localization, the cytoplasmic and nuclear proteins of the human liver cancer cell line HepG2 were isolated for the detection of Vasohibin-2 by western blotting. Vasohibin-2 cDNA, coding for 311 and 355 amino acid residues, fused with or without a DDK/V5 tag at the c-terminus, respectively, was cloned into the Lv-CMV-EGFP vector. Lentiviruses were successfully packaged. Vasohibin-2-overexpressing HepG2-VASH2 (355 amino acid residues) and HepG2-VASH2-V5 (311 amino acid residues fused with V5 tag at the c-terminus) human liver cancer cell lines were established. Approximately 1-2x106 HepG2, HepG2-VASH2 and HepG2-VASH2-V5 cells were injected subcutaneously into the flanks of BALB/c nude mice. Xenograft tumors were harvested for immunohistochemistry. HepG2 cells were transiently transfected with the Lv-CMV-EGFP vectors containing Vasohibin-2 cDNA (coding for 311/355 amino acid residues with a DDK tag at the c-terminal), followed by anti-DDK immunofluorescence. The antibodies obtained were able to detect human VASH2 successfully as applied in western blotting, IF, IHC and IP. Results from IF, IHC and WB (post cytoplasmic/nuclear protein isolation) showed a quite different intracellular localization of VASH2 protein. The VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. The former was found to be a relatively low abundance protein. We successfully generated three rabbit anti-human Vasohibin-2 polyclonal antibodies which can be used for western blotting, IF, IP and IHC. These antibodies will provide a convenient tool for further studies on Vasohibin-2. This is the first study to report differences in the intracellular localization of the VASH2 protein and, hence, a new research direction on the study of VASH2.

Vasohibin 2 reduces chemosensitivity to gemcitabine in pancreatic cancer cells via JUN proto-oncogene dependent transactivation of ribonucleotide reductase regulatory subunit M2

“…VASH2 is a homolog of VASH1, and was shown to stimulate angiogenesis in a mouse model of hypoxia-induced subcutaneous angiogenesis [3]. We have demonstrated that VASH2 protein expression can be detected in both the nuclear and cytoplasmic compartments [4]. Recently, VASH2 has been demonstrated to be involved in the malignant behavior of a number of malignancies, including hepatic cancer [5,6], ovarian cancer [7,8], endometrial cancer [9], gastrointestinal cancers [10], breast cancer [11], and pancreatic cancer [12].…”

“…Whole cell lysates were prepared in radioimmunoprecipitation assay buffer (Beyotime, Nantong, China) and blotted using the following primary antibodies: rabbit polyclonal anti-VASH2 (prepared as described in [4]); rabbit polyclonal anti-RRM2 (cat. no.…”

“…We previously produced rabbit polyclonal anti-human VASH2 antibodies which were successfully applied in immunoblotting and immunohistochemical analyses of human liver cancer and adjacent normal tissues [4,13], breast cancer [11], and multiple other human cancer and normal tissues [14]. Here, we investigated the expression of VASH2 in human pancreatic cancer and analyzed the relationship between VASH2 expression and clinical features.…”

Vasohibin-2 promotes proliferation in human breast cancer cells via upregulation of fibroblast growth factor-2 and growth/differentiation factor-15 expression

“…It was previously demonstrated that there are two types of VASH2: Nuclear and cytoplasmic (7). In the present study, the focus was on cytoplasmic VASH2, thus all subsequent mention of VASH2 refers to the cytoplasmic form.…”

“…In the present study, the focus was on cytoplasmic VASH2, thus all subsequent mention of VASH2 refers to the cytoplasmic form. VASH2 expression was investigated in human breast cancer in the current study; rabbit polyclonal anti-human VASH2 antibodies were produced and successfully used in immunoblotting and immunohistochemical analysis (7). …”