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H-J Suk, JH Sohn, HJ Kaplan, PS Bora, NS Bora; Clusterin, a Complement Regulatory Protein, is Present in Normal Rat Eye . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2283.
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Purpose: We have previously shown that Crry and CD59 are present in the normal rat eye. The present study was undertaken to detect the presence of clusterin-another complement regulatory protein - in the eye of the normal Lewis rat. Method: Aqueous humor was obtained from normal Lewis rat eyes. Soluble ocular extract was prepared by homogenizing and solubilizing the intraocular content of normal rat eyes in PBS containing protease inhibitors. Immunoblotting was performed using the enhanced chemiluminescence Western blotting detection system "ECL + Plus" (Amersham Pharmacia Biotech, Arlington Heights, IL). Briefly, electrophoresis was performed on a 12% SDS-PAGE slab gel under non-reducing conditions and the separated proteins were transferred to PVDF membranes. The blot was then reacted with 1µg/ml of monoclonal anti-clusterin antibody(7A8-E4) or the same concentration of MOPC-21 and UPC10, separately, overnight at 4ºC. The bound antibodies were visualized by using goat anti-mouse IgG that had been conjugated with horseradish peroxidase (HRP). Results: On Western blot analysis clusterin appeared as a single band of 40 kDa in normal rat aqueous humor, as well as in the soluble extract obtained from the normal rat eye. No signal was obtained in control blots incubated with MOPC-21 and UPC-10. Conclusion: Our results suggest that clusterin, a regulator of membrane attack complex (MAC) of complement , is present in normal rat aqueous humor and intraocular tissue. Thus, a complex immunoregulatory complement network exists within the eye presumably to prevent inadvertent bystander tissue damage during innate immunity.
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