Saturation BRET analysis supported the specific interaction of Lrp5 and GCG

edium was collected, centrifuged to remove dead cells and debris and GSH and/or GSSG release was determined in the cell-free medium. Total protein was isolated from the cells, quantified and intracellular GSH or GSSG content was measured. GSH release was expressed as nmol/ml per unit time. Generation of stable cell lines In order to ensure consistency in transfection studies, stable transfections were performed in ARPE-19 cells. Cells were transfected with the neomycinresistant pcDNA vectors containing aA or aB crystallin inserts using FuGene 6 transfection reagent. Cells were allowed to recover in DMEM/HAM’s F12 with 10% FBS for 24 h and were sub-cultured in selection medium containing 500 mg/ml G418 sulfate. After 3 weeks, individual colonies were isolated, subcultured, expanded and examined for expression of aA and aB crystallin by immunoblot analysis with anti-aA and anti-aB crystallin antibodies. Immunoblot analysis Cells were harvested after the specified treatment period and protein was extracted from the cells or posterior eye cups. Equal amounts of protein were resolved on 15 or 4 15% Tris-HCl polyacrylamide gels as described previously. Membranes were probed with 19782727 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 9336340 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 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- BCTC 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 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 ice for 30 min with vortexing in between every 5 min. The samples were centrifuged and the supernatant was added to the microcentrifuge spin col