C. tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained and on the type of specimens (cell, tissue/cell homogenate or section) tested. Keywords: selective antibodies, alpha adrenergic receptors, vasopressin receptor, chemokine receptor Introduction G protein-coupled receptors (GPCR) or 7 transmembrane domain name (7TM) receptors are the largest group of eukaryotic cell surface receptors and play important functions in physiology and pathology (Alexander et al., 2013, Venkatakrishnan et al., 2013, Vaidehi et al., 2014). Nevirapine (Viramune) It has been estimated that approximately 50% of all currently available drugs target GPCRs (Fredriksson and Schioth, 2005). Thus, selective antibodies that detect endogenous GPCRs can be essential for the study of these receptors (Gupta and Devi, 2006, Talmont et al., 2012). Several lines of evidence, however, suggested that many commercially available antibodies directed against GPCRs, such as histamine receptors, adrenoceptors or chemokine receptors, lack sufficient selectivity (Hamdani and van der Velden, Nevirapine (Viramune) 2009, Jensen et al., 2009, Pradidarcheep et al., 2009, Berahovich et al., 2010, Beermann et al., 2012, Bohmer et al., 2014, Cecyre et al., 2014, Cernecka et al., 2014, Talmont and Mouledous, 2014). Accordingly, it has been proposed that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down methods such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell collection and/or d) antibodies raised against multiple unique epitopes of a receptor yielding very similar staining patterns (Michel et al., 2009). We have shown previously that several commercially available antibodies against chemokine (C-X-C motif) receptor 4 (CXCR4) show acceptable selectivity as we observed a major reduction of staining with these antibodies when endogenous CXCR4 was silenced with siRNA (Saini et al., 2010, Tripathi et al., 2015). In the present study, we again applied these criteria and tested twelve commercially available antibodies directed against GPCR targets that are in the center of our current research interests: -adrenoceptors, atypical chemokine receptor 3 (ACKR3) and vasopressin receptor 1A (AVPR1A). The tested antibodies included two antibodies that have previously been reported to be non-specific and an antibody that has been thoroughly evaluated and found to be specific for the GPCR target (Jensen et al., 2009, Berahovich et al., 2010). Materials and Methods Cells and reagents Human aortic vascular easy muscle mass cells (hVSMC) were obtained from American Type Culture Collection (ATCC) and cultured as Nevirapine (Viramune) explained in detail previously (Tripathi et al., 2015). Antibodies were obtained from commercial sources and are outlined in Table 1. siRNA reagents were purchased from Thermo Scientific Dharmacon. Table 1 Antibodies tested

abcamab1371231A-ARRabbit mAbFlow Cyt, ICC/IF, WBHu, Ms, RtHuman ADRA1A (148) (E-005419-00-0050)62 13 (8)Santa Cruzsc14771A-ARGoat pAbWB, IF, ELISAHu, Ms, Rt, CowHuman ADRA1A (148) (E-005419-00-0050)70 19 (4)abcamab1695231B-ARRabbit mAbFlow Cyt, ICC/IF, WBHu, Ms, RtHuman ADRA1B (147) (E-005420-00-0050)82 19 (8)abcamab844021D-ARRabbit pAbWB, IHC-PHu, Ms, Rt, Dm, ZfHuman ADRA1D (146) (E-005421-00-0050)74 22 (7)Santa Cruzsc270991D-ARGoat pAbWB, IF, ELISAHuHuman ADRA1D (146) (E-005421-00-0050)85 3 (4)Sigmasab45005482A-ARRabbit pAbIHC, WBHu, Ms, RtHuman ADRA2A (150) (E-005422-00-0050)46 10 (4)abcamab217682B-ARRabbit mAbFlow Cyt, WB, IPHu, Ms, RtHuman ADRA2B (151) (E-005423-00-0050)60 2 (4)abcamab1674332C-ARMouse mAbWB, ICCHu, Ms, Rt, GpHuman ADRA2C (152) (E-005424-00-0050)70 20 (3)R&D Systemsmab42273 clone 11G8ACKR3Mouse mAbFlow Cyt, IHCHuHuman CXCR7 (57007) (E-013212-00-0050)72 5 (3)abcamab38089ACKR3Rabbit pAbIHC-P, WBHu, MsHuman CXCR7 (57007) (E-013212-00-0050)80 4 (3)LSBioLS-B1815ACKR3Rabbit pAbIHC-P, WB, Circulation.