He two kinds of receptors preparations were used to immunize animals. BALB/c mice were injected subcutaneously with 100 mg of purified receptors emulsified in complete Freund’s adjuvant followed by two injections two weeks apart with the same amounts of proteins in incomplete Freund’s adjuvant. For each GPCR preparation (i.e. in water and in SDS), two sets of immunization were performed on three or four animals.Detection of receptors expressed in recombinant cellsThe antibody specificity of serum IgG collected from immunized mice was first examined by western-blotting on the wild-type recombinant receptors without c-myc tag fused to the C terminus. The ability 12926553 of polyclonal buy 4 IBP antibodies (serum dilution ranging from 1/500 to 1/4000) to specifically recognize receptors was assessed by comparing their immunodetection in extracts from membrane of CHO-K1 cells expressing the relevant GPCR (1?0 pmol/mg membrane proteins) and from wild-type CHO-K1 cells. For each receptor, a unique band was revealed by immune serum IgG antibodies as assessed by western-blotting (Fig. 2a). Similar resultsResults Immunogen preparation and immunizationRecombinant human G-protein coupled receptors with six histidine residues and a c-myc tag fused to their C-terminus were produced in the methylotrophic yeast Pichia Pastoris. Receptors were solubilized in 0.1 sodium dodecyl sulphate (SDS) and 8 M urea and subsequently chromatographed upon nickel affinity column. Receptors that bound to nickel-agarose phase because ofAntibodies against G-Protein Coupled ReceptorsTable 1. Characteristics of human G-protein coupled receptors used to generate immune serum IgG antibodies.Receptor hMOR hKOR hNPFFRGene OPRM1 OPRK1 NPFFAccession number NP 000905 NP 000903 NP 444264.Size 
(AA) 400 380Theoretical Molecular weight (kDa) 44.78 42.65 48.AA: amino acids. kDa: kilodalton. doi:10.1371/journal.pone.0046348.twere obtained with all individual immune sera from mice immunized with GPCRs both in water and 0.1 SDS. No IgG binding to control CHO-K1 cell membranes was observed. The apparent molecular weights of all the three receptors, revealed by immune sera as a 
unique band, were higher than theoretical ones or those observed when receptors originated from yeast. Bands were observed respectively at 80 kDa, 60 kDa and 70 kDa for hNPFFR2, hKOR and hMOR expressed on CHO cell membranes while their theoretical molecular weights calculated from the standard atomic weights are 49 kDa, 43 kDa and 45 kDa (Table 1). The discrepancy between the theoretical molecular weights of the receptors and the molecular weights corresponding to the bands revealed by anti-GPCR antibodies suggested that the receptors were probably glycosylated in CHO mammalian cells, as already described for many other 15755315 GPCRs [32,33]. This assumption was validated by deglycosylating the hNPFFR2 receptor with Peptide N Glycosidase F, which cleaves asparagine-linked oligosaccharides from glycoproteins, prior ASP015K assessing it by western-blotting. As shown in figure 3a, anti-hNPFFR2 IgG antibodies revealed, in addition to the band at 80 kDa, others bands with lower apparent molecular weights. Thus, as exemplified for hNPFFR2, anti-GPCR polyclonal antibodies may recognize receptors with and without N-glycans. Anti-GPCR immune sera were also able to recognize receptors in their native conformation at the membrane surface of CHO cells as assessed by confocal microscopy (Fig. 2b) and cytofluorometry (Fig. 2c). Each immune serum IgG stained CHO.He two kinds of receptors preparations were used to immunize animals. BALB/c mice were injected subcutaneously with 100 mg of purified receptors emulsified in complete Freund’s adjuvant followed by two injections two weeks apart with the same amounts of proteins in incomplete Freund’s adjuvant. For each GPCR preparation (i.e. in water and in SDS), two sets of immunization were performed on three or four animals.Detection of receptors expressed in recombinant cellsThe antibody specificity of serum IgG collected from immunized mice was first examined by western-blotting on the wild-type recombinant receptors without c-myc tag fused to the C terminus. The ability 12926553 of polyclonal antibodies (serum dilution ranging from 1/500 to 1/4000) to specifically recognize receptors was assessed by comparing their immunodetection in extracts from membrane of CHO-K1 cells expressing the relevant GPCR (1?0 pmol/mg membrane proteins) and from wild-type CHO-K1 cells. For each receptor, a unique band was revealed by immune serum IgG antibodies as assessed by western-blotting (Fig. 2a). Similar resultsResults Immunogen preparation and immunizationRecombinant human G-protein coupled receptors with six histidine residues and a c-myc tag fused to their C-terminus were produced in the methylotrophic yeast Pichia Pastoris. Receptors were solubilized in 0.1 sodium dodecyl sulphate (SDS) and 8 M urea and subsequently chromatographed upon nickel affinity column. Receptors that bound to nickel-agarose phase because ofAntibodies against G-Protein Coupled ReceptorsTable 1. Characteristics of human G-protein coupled receptors used to generate immune serum IgG antibodies.Receptor hMOR hKOR hNPFFRGene OPRM1 OPRK1 NPFFAccession number NP 000905 NP 000903 NP 444264.Size (AA) 400 380Theoretical Molecular weight (kDa) 44.78 42.65 48.AA: amino acids. kDa: kilodalton. doi:10.1371/journal.pone.0046348.twere obtained with all individual immune sera from mice immunized with GPCRs both in water and 0.1 SDS. No IgG binding to control CHO-K1 cell membranes was observed. The apparent molecular weights of all the three receptors, revealed by immune sera as a unique band, were higher than theoretical ones or those observed when receptors originated from yeast. Bands were observed respectively at 80 kDa, 60 kDa and 70 kDa for hNPFFR2, hKOR and hMOR expressed on CHO cell membranes while their theoretical molecular weights calculated from the standard atomic weights are 49 kDa, 43 kDa and 45 kDa (Table 1). The discrepancy between the theoretical molecular weights of the receptors and the molecular weights corresponding to the bands revealed by anti-GPCR antibodies suggested that the receptors were probably glycosylated in CHO mammalian cells, as already described for many other 15755315 GPCRs [32,33]. This assumption was validated by deglycosylating the hNPFFR2 receptor with Peptide N Glycosidase F, which cleaves asparagine-linked oligosaccharides from glycoproteins, prior assessing it by western-blotting. As shown in figure 3a, anti-hNPFFR2 IgG antibodies revealed, in addition to the band at 80 kDa, others bands with lower apparent molecular weights. Thus, as exemplified for hNPFFR2, anti-GPCR polyclonal antibodies may recognize receptors with and without N-glycans. Anti-GPCR immune sera were also able to recognize receptors in their native conformation at the membrane surface of CHO cells as assessed by confocal microscopy (Fig. 2b) and cytofluorometry (Fig. 2c). Each immune serum IgG stained CHO.
