The Kaplan-Meier method was used for comparison of the tumor development induced by DMBA/TPA

described previously. Membranes were probed with rabbit polyclonal glutamatecysteine ligase, catalytic subunit , polyclonal glutamate-cysteine ligase, modifier subunit anti-MRP1, anti-glutathione reductase, anti-aA crystallin, anti-aB crystallin, overnight at 4uC. After incubation with the corresponding secondary antibodies, signals were detected using an enhanced chemiluminescence system, membranes reprobed for GAPDH or b-actin. MRP1 overexpression Generation of the human MRP1 cDNA cloned into the pcDNA 3.1 vector has been described. ARPE-19 cells were transfected with the MRP1 pcDNA 3.1 vector and 48 h after transfection, mRNA and protein was isolated. Expression of MRP1 in the transfected cells was determined by real-time RTPCR and by immunoblot analysis using a mouse monoclonal MRP1 antibody. Cellular toxicity was determined by LDH assay. Quantitative real-time PCR MRP1-Mediated GSH Efflux in RPE Cells calculating 22DDCT. Results are reported as mean difference in relative multiples of change in mRNA expression 6 SEM. Immunofluorescence cell staining Cells were grown on 4-well chamber slides or human fetal RPE monolayers on transwell filters were processed. After incubation with primary antibody, slides were incubated with fluorescein -conjugated secondary antibody and were examined using a laser scanning confocal purchase GSK-126 microscope. protein were extracted from the posterior eye cup. Real-time PCR was used to amplify the mRNA levels. Data are normalized to L32 and presented as relative fold difference over control. 2550 mg total protein was loaded for Western blot analysis and probed with rabbit Trx1, goat Trx2 and rabbit Grx1. GAPDH was used as a loading control. All four redox proteins showed a significant decrease in expression when compared to corresponding age-matched wild type. Trx1- Thioredoxin 1, Trx2- Thioredoxin 2, Grx1- Glutaredoxin 1, Grx2- Glutaredoxin 2. P,0.05, P,0.01. Biotinylation RPE cells at 90% confluence were used for biotinylation as suggested by the manufacturer. Briefly, cells were incubated with 10411607” 10 ml biotin solution on a shaker for 30 min at 4uC and the cells were gently scraped and collected by centrifugation. The cells were sonicated and incubated on 10525069” ice for 30 min with vortexing in between every 5 min. The samples were centrifuged and the supernatant was added to the microcentrifuge spin column. The column was subjected to low speed centrifugation, and finally 300 ml of sample buffer was added to the column and incubated 1 hr at room temperature. The membrane fraction was collected by centrifugation and was subjected to immunoblot analysis. Data Analysis Data were analyzed with InStat. ANOVA and Tukey post hoc test were used to assess the differences between groups. P,0.05 was considered to be statistically significant. Acknowledgments We wish to thank Dr. V. Ganapathy, Medical College of Georgia for helpful discussions. Hepatitis B is a public health problem worldwide. As estimated, two billion people have been infected with HBV. The subviral particles of HBV are produced in vast excess during the life cycle of the virus, whose concentrations could reach 50300 mg/ml in blood. HBV is able not only to pass through the blood-testis barrier and enter the male germ cells but also integrate into their genomes.The previous work has confirmed that human sperm cells could serve as possible vectors for vertical transmission of HBV genes. After being introduced into the embryo via the sperm, HBV genes were replicated an