Intracellular GSH levels in MRP1 overexpressing cells were significantly lower than vector control cells

The procedures for isolating primary hepatocytes have been described previously. Western blot BAY41-2272 analysis HEK293 or other cells were cultured in 6-well plates. For experiments requiring transfection, cells were transfected with the indicated DNA using Lipofectamine 2000 according to the manufacturer’s protocol. After transfection, cells were treated with different reagents for the indicated time. Cell pellets were lysed in 90 ml of 16 cell lysis buffer on ice for 30 min and western blots were performed as described earlier. The blots were developed with Pico Chemiluminescence substrate. After 24 h, the cells were transiently transfected with 3 mg of DNA/dish using the diethylaminoethyl -dextran method. Cells were used 4872 h later. Receptor-bearing COS-1 cell suspensions of approximately 25,000 cells/well were used for bioluminescence and fluorescence measurements in 96-well Optiplates. BRET Glucagon Induced b-Catenin Signaling Pathway assays were initiated by mixing 5 mM coelenterazine h with the cell suspension. The luminescence signals were collected immediately using a 2103 Envision fluorescence plate reader configured with the,700 nm dichroic mirror and with dual emission filter sets for luminescence and fluorescence. Fluorescence of the YFP was acquired by exciting the samples at 485 nm and collecting the emission at 525 nm. The BRET ratios were calculated based on the ratio of emission from YFP and Rlu, as described previously. Saturation BRET studies were also performed as described previously. In brief, COS-1 cells were transfected with a fixed concentration of Rlu-tagged constructs as donor and with increasing concentrations of YFP-tagged constructs as acceptors. After 4872 h, BRET assays were performed. The BRET signals were plotted as ratios relative to the ratios of emissions of YFP/Rlu, and the curve fit was evaluated based on R2 values using Prism 4.0.. 1 and 3, C-terminal region of Frizzled receptors and three class B GPCRs. The IC loops and C-terminal region were predicted by the HMMTOP server and aligned by clustalW program. The conserved residues critical for activation of Wnt/b-catenin signaling are highlighted in yellow based on previous studies. Single mutations abolish Wnt/bcatenin signaling activity of human Frizzled 5 are indicated on the top of the alignment. Residue number corresponds to human Fz5 sequence. Supporting Information Acknowledgments We thank Jiandie Lin and Matthew Molusky at the University of Michigan for providing us with help in isolating primary liver cells from ” mice. We thank Alicja M. Ball and Mary-Lou Augustine for assistance with cell culture for the BRET studies. In addition, we thank David Nadziejka for editorial assistance in preparing the manuscript. Coexpression of Lrp5 enhanced the CRE Luciferase activity. HEK293 cells were transfected with GCGR or GCGRLrp5 plasmids along with CRE-Luc and TKRlu on day 1. Cells were left untreated or treated with 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the luciferase activity as described. After initial clinical descriptions, mutations in the alphagalactosidase A gene were found to be responsible for Fabry disease, which is an X-linked disorder of glycosphingolipid metabolism that results in progressive accumulation of neutral glycosphingolipids, in ” lysosomes, as well as other cellular compartments and the extracellular space. The prevalence of Fabry mutation ranges from 1 in 40,000 to 1:117,000 in United States and Australia