FIGURE 1.

OR51E2 is expressed in human epidermal melanocytes. A, RT-PCR detection of 13 different ORs in normal human epidermal melanocytes (NHEM). Gel electrophoresis of OR amplicons from melanocyte cDNA (+) and no reverse transcriptase cDNA controls (−) to exclude genomic DNA contamination is shown. Amplification of actin and tyrosinase-related protein 1 (Tyr-P1) served as positive controls. OR51B5, OR5P3, and OR6B3 transcripts were not detected. B, detection of OR51E2 transcripts in melanocytes (normal human epidermal melanocytes) by RT-PCR together with a schematic representation of the intron over-spanning primers for this receptor. OR51E2 transcripts could be detected in melanocytes of different pigmentation levels (Caucasian and Negroid origin). LNCaP cells served as the positive control and negative control: genomic DNA (gDNA). The size of the OR51E2 amplicon was 1050 bp (cDNA) or >15 000 bp (gDNA). C, detection of OR51E2 protein in primary melanocytes by Western blotting; the size of the OR51E2 monomeric (51E2-M) protein was 35 kDa and dimeric protein was 70 kDa (51E2-D). D, immunocytochemical staining using an OR51E2-specific antibody; shown are confocal micrographs of an OR51E2 staining in melanocytes. The control consisted of omission of the primary antibody. Bars indicate 15 μm. E, co-immunocytochemical staining of Hana3a cells transiently expressing OR51E2 with an N-terminal rho tag, which consists of the first 20 amino acids of rhodopsin. Detection of the recombinant rho-OR51E2- protein was performed using an antibody against OR51E2 and an antibody against the N-terminal rho tag. Shown are confocal micrographs of the OR51E2 staining in OR51E2 expressing Hana3a cells. Mock-transfected Hana3a cells served as negative control. Bars indicate 20 μm. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Co-labeling (yellow) of OR51E2-expressing Hana3a cells indicates specificity of the used OR51E2 antibody.