Department of Chemistry and Molecular Biology and the Center for Protease Research, North Dakota State University, Fargo, ND 58108-6050, United States.
J Immunol Methods. 2012 Feb 28;376(1-2):20-31. doi: 10.1016/j.jim.2011.10.009. Epub 2011 Nov 4.
Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.
血管活性肠肽受体-1（VPAC1）信号在淋巴细胞中被证明可以调节趋化性、增殖、凋亡和分化。在 T 细胞激活过程中，VPAC1mRNA 下调，但对其蛋白水平的影响尚不清楚。少数研究报道了使用流式细胞术测量人 VPAC1，但鼠 VPAC1 试剂不可用。因此，我们着手生成一种可靠且高度特异性的α-鼠 VPAC1 多克隆抗体，用于流式细胞术。在成功生成兔α-VPAC1 多克隆抗体（α-mVPAC1 pAb）后，我们对其交叉反应性进行了表征，并表明它不能通过流式细胞术识别其他家族受体（鼠 VPAC2 和 PAC1 以及人 VPAC1、VPAC2 和 PAC1）。兔α-VPAC1 血清的部分纯化将α-mVPAC1 pAb 的比活性提高了 20 倍，免疫荧光显微镜（IF）证实了鼠 VPAC1 蛋白的细胞膜亚细胞定位。为了测试这种特异性α-mVPAC1 pAb 的有用性，我们表明原代、静止的鼠 T 细胞表达可检测水平的 VPAC1 蛋白，而活化的 T 细胞或 CD19 B 细胞的信号很少可检测到。这些数据支持我们之前发表的数据，表明在 T 细胞激活过程中 VPAC1mRNA 下调。总的来说，我们已经建立了一种经过充分表征的、高度种属特异性的α-mVPAC1 pAb，可用于 IF 和流式细胞术测量 VPAC1 表面。
