Immunolocalization of X-arrestin in human red/green cone photoreceptors

Purpose. X-arrestin is a recently identified retina-specific gene of unknown function. To begin to probe the function of this gene, we investigated its cellular localization in human retina using an antibody against X-arrestin. Methods. Affinity-purified polyclonal anti-peptide antibody to human X-arrestin was prepared, its specificity was confirmed by Western blot analysis, and the antibody was used for immunofluorescent staining of human retinal sections. Anti-human red/green cone opsin antibody, anti-bovine arrestin (S-Ag) antibody, and anti-bovine rhodopsin antibody were also used for comparison. Results. The anti-peptide antibody showed specific activity against X-arrestin with an apparent molecular mass of ∼47 kDa by Western blot analysis. In the microscopic sections, strong immunoreactivity was observed exclusively in the cones, with greatest intensity in the cone outer segments. An identical pattern of immunolabeling was observed in cones following double staining with the X-arrestin and red/green cone opsin antibodies. As predicted, very different patterns of immunostaining were obtained with the S-antigen and rhodopsin antibodies, which specifically stained the rods. A double immunofluorescent analysis with the X-arrestin and S-antigen antibodies clearly demonstrated the distinct staining of the cones and rods, respectively. Conclusions. The results showed that our anti-peptide antibody is specific for X-arrestin and that X-arrestin is specifically localized to human red/green cone photoreceptors.