There small bands of both BCO2 and VDAC1 in the nuclear fraction (Fig. ability to store vitamin A. Liver is also known to accumulate carotenoids, however, their uptake, retention and metabolism in specific liver and intestinal cell Quinfamide (WIN-40014) types is still unknown. Hence, we studied the cellular and subcellular expression and localization of BCO1 and BCO2 proteins in rat liver and intestine. We demonstrate that both BCO1 and BCO2 proteins Quinfamide (WIN-40014) are localized in hepatocytes and Quinfamide (WIN-40014) mucosal epithelium. We also show that BCO1 is also highly expressed in hepatic stellate cells (HSC) and portal endothelial cells in liver. At the subcellular level in liver, BCO1 is found in cytosol, while BCO2 is found in mitochondria. In intestine, immunohistochemistry showed strong BCO1 immunoreactivity in the duodenum, particularly in Brunners glands. Both BCO1 and BCO2 showed diffuse presence along epithelia with strong immunoreactivity in endothelial cells and in certain epithelial cells which warrant further investigation as possible intestinal retinoid storage cells. and models have shown that both hepatocytes and hepatic stellate cells accumulate carotenoid [19C21]. Hepatic accumulation of -carotene has been observed in both the and mRNA levels in primary hepatic stellate cells (HSC) as compared to isolated primary hepatocytes in mice [20]. Additionally, while earlier studies have reported a cytoplasmic localization of BCO2 in cell, two recent studies demonstrate that BCO2 is a mitochondrial protein [22,26]. Greater understanding of the localization of carotenoid cleavage enzymes is needed to better understand the metabolism of carotenoids in liver and intestine. Hence, we studied the cellular and subcellular expression and localization of BCO1 and BCO2 proteins in rat liver and intestine. We demonstrate that both BCO1 and BCO2 proteins are localized in hepatocytes and mucosal epithelium. We also show that BCO1 is also highly expressed in hepatic stellate cells (HSC) and portal endothelial cells in liver. At the subcellular level in liver, BCO1 is found in cytosol, while BCO2 is found in mitochondria. In intestine, immunohistochemistry showed strong BCO1 immunoreactivity in the duodenum, particularly in Brunners glands. Both BCO1 and BCO2 showed diffuse presence along epithelia with strong immunoreactivity in endothelial cells and in certain epithelial cells which warrant further investigation as possible intestinal retinoid storage cells. Materials and methods Cell lines Rat hepatoma McArdle RH7777 (McA) cell line (ATCC, #CRL-1601) was maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (SigmaCAldrich, St Louis, MO), 100 units penicillin and 100 g streptomycin/ml (Invitrogen #15140), and 0.2 ml/100 ml Fungizone (Invitrogen #15290). Chinese hamster ovary (CHO) cells, stably transfected with mouse cDNA and referred as NY CHO cells in this study, were used as a positive control in assessing the specificity of the antibody for BCO1 protein. The non-transfected chinese hamster ovary cell line, obtained Rabbit polyclonal to APEH from American Type Culture Collection (ATCC, #CRL-10154), was referred to as ATCC CHO cells and was used as a negative control. NY CHO and ATCC CHO cells were maintained in basic medium, MEM (+10% FBS, 100 units penicillin/streptomycin/ml, and 0.2 ml/100 ml Fungizone). All the cell lines were incubated at 37 C with 5% Quinfamide (WIN-40014) CO2 and 95% humidity. All chemicals were purchased from Invitrogen or as specified. Antibodies A rabbit polyclonal antibody against mouse BCO1 (IgG) referred to as BCO1 antibody, was prepared as described [14]. A chicken anti BCO2 was prepared using a synthetic peptide unique to the C-terminus of human BCO2 (Genway biotech, San Diego, CA). A commercially-available rabbit antibody against human desmin (Novus Biological, Littleton, CO) was used as marker for hepatic stellate cells. Polyclonal rabbit anti-VDAC1 antibody was used as mitochondrial marker for immunostaining of Western blots. Secondary antibodies used in immunohistochemistry (IHC) were; biotinCstreptavidin-conjugated donkey anti-rabbit IgG and anti-chicken IgY (Jackson Immunoresearch, West Grove, PA). Peroxidase conjugated mouse anti-biotin IgG (Jackson Immunoresearch) was used as tertiary antibody. For Western blotting, secondary antibodies used were infrared dye conjugated, donkey anti-chicken and goat.