These data suggest that the -mVPAC1 pAb is species specific as it is unable to recognize functionally active, recombinant or endogenously expressed human VPAC1 protein by circulation cytometry, and therefore can distinguish between mouse and human VPAC1 protein. == Physique 2. -VPAC1 sera increased the specific-activity of the -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 -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 -mVPAC1 pAb for VPAC1 surface measurement by IF and circulation cytometry. Keywords:T cells, vasoactive intestinal peptide receptor 1, vasoactive intestinal peptide receptor 2, pituitary Adenylate Cyclase activating polypeptide 1, polyclonal antibody, circulation cytometry == 1. Introduction == Vasoactive intestinal peptide/pituitaryadenylatecyclase activating polypeptide receptor 1 (VPAC1) is the prototypical, group II, G protein coupled receptor nearly ubiquitously expressed in mammals (Ceraudo et al., 2008). This receptor binds the neuropeptide called vasoactive intestinal peptide (VIP) with 1 nM Kd(Gaudin et al., 1996), and its neuronal source is derived from neurons in the central nervous system (Lorn et al., 1979), and peripheral neurons that deliver this ligand to numerous organs (Felten et al., 1985;Ottaway et al., 1987). Also, VIP is usually expressed by certain immune cells, including activated CD4 Th2cells (Danek et al., 1983;Delgado and Ganea, 2001). The transmission transduction pathways elicited by VIP/VPAC1 are through differential Rabbit Polyclonal to ABCA8 coupling between Gs, Giand Gqthat appear to be cell context dependent (Van Rampelbergh et al., 1997;McCulloch et al., 2002). Major cellular effects for the VIP/VPAC1 signaling axis are to regulate electrolyte balance (Schwartz et al., 1974), secretion of soluble mediators (Delgado et al., 1999), proliferation (Rameshwar et al., 2002a), chemotaxis (Johnston et al., 1994) and apoptosis (Delgado and Ganea, 2000). Several disease states have been implicated with abnormal VPAC1 levels, including inflammatory disorders such as Rheumatoid arthritis (Woessner, 1991;Yuko et Btk inhibitor 1 al., 1999) and inflammatory bowel disease (Abad et al., 2003), and solid cancers originating from the lung, prostate, breast, and brain (Reubi, 1996;Reubi et al., 2000a). The VPAC1 gene was first cloned in 1992 using a rat lung cDNA library, followed by the cloning of the human VPAC1 gene in 1995, which made an immediate impact regarding the tissue expression profile exposing high expression in lung, liver, and intestines (Ishihara et al., 1992;Sreedharan et al., 1995). A decade later, many antibodies had been developed using peptide sequences from your amino- and carboxyl-terminal regions of VPAC1, as well Btk inhibitor 1 as, the entire full-length human VPAC1 sequence (Goetzl et al., 1994a;Schulz et al., 2004;Freson et al., 2008). These antibodies proved invaluable for detecting VPAC1 protein expression by Btk inhibitor 1 end-point analyses including, immuno blots, immunoprecipitation, immunohistochemistry and immunofluorescence (Goetzl et al., 1994b;Jiang et al., 1998;Schulz et al., 2004;Zhang et al., 2008). Interestingly, you will find exceedingly few examples of circulation cytometry analyses using antibodies to the VPAC receptors, and of these studies that we are aware of, only human VPAC protein was measured (Marie et al., 1999;Langlet et al., 2005;Sun et al., 2006;Delgado et al., 2008;Freson et al., 2008). Indeed, we as well as others have relied primarily on RT-PCR, andin situhybridization to measure mouse VPAC1 expression at the mRNA level (Barberi et al., 2007; Dorsam, 2010). Therefore, the need for any species, specific, mouse VPAC1 antibody suitable for circulation cytometry would be a useful resource for the neuroimmunology field. Some success has, however, been reported using fluorescently conjugated VIP ligands that measured the presence of binding sites on cells (Lara-Marquez et al., 2001b), but this strategy does not distinguish between VPAC1 and a 50% homologous VPAC2 receptor (Igarashi et al., 2002). Radioactively iodinated VIP/PACAP ligand binding assays have also become a routine method for discerning between the non-selective VPAC1 and VPAC2 receptorsversesthe selective pituitary adenylate cyclase activating polypeptide receptor 1 (PAC1) (Vertongen et al., 1997;Reubi et al., 2000b). However, this strategy is dependent on proper acknowledgement of the.