Abstract
Abstract: :
Purpose: To determine the intracellular localization of several OSBPs present in ARPE19 cells and their translocation in response to internalization of LDL, oxLDL and oxysterols. Methods: ARPE19 cells were transfected with the BD Living Colors Subcellular Localization Vectors – ECFP and DsRed2 (Clontech, Palo Alto, CA) using Cell Line Nucleofector Kit V and a Nucleofector I instrument (Amaxa Biosystems, Koeln, Germany). Immunocytochemistry was performed on transfected ARPE19 cells plated on 8 well chamber slides and fixed in 4% fresh paraformaldehyde –PBS for 10 min. Antibodies to OSBP1, 2, 4 and 6 were affinity purified. A biotin–labled anti–rabbit was used as secondary antibody in combination with CY3 Streptavidin (KPL, Gaithersburg,MD) or AlexaFluor 488 FluoroNanogold Streptavidin (Molecular Probes, Eugene,OR) The nuclei were counter–stained with DAPI. The cells were examined by confocal and light fluorescent microscopy. Results: In the ARPE19 cells, endogenous OSBP1 associated predominantly with the Golgi apparatus. OSBP2 and OSBP6 were distributed between the nucleus and cytosol, OSBP4 was found in the cytosol and partially associated with ER. None of the OSBPs showed clear translocation in response to LDL or oxLDL internalization within 24 hrs. Conclusions:All of the OSBPs examined showed distinct intracellular localizations. Preliminary results demonstrated no obvious translocation of the OSBPs after internalization of LDL or oxLDL. These results require further validation and more detailed experimentation using OSBP–GFP expression constructs is in progress.
Keywords: age–related macular degeneration • immunohistochemistry • retinal pigment epithelium