The mouse hypothalamic cell line GT1-7 was a gift from Dr. Pam Mellon (UCSD). GT1-7 cells, 293T cells and GH3 cells were cultured in DMEM (Mediatech) containing 10% fetal bovine serum (HyClone), 100 μg/ml penicillin-streptomycin and 1mM pyruvate. Transient transfections of GT1-7 cells were performed using Fugene HD (Roche). HEK293T cells and GH3 cells were transfected by using Lipofectamine 2000 (Invitrogen). For luciferase assays, cells were seeded in 24-well plates and transfected 16h later. Cells were treated for 4h, as indicated, 16 to 48h post-transfection. In reporter assays employing LRb, cells were transfected and treated under serum-free conditions. Cell lysates were prepared and the activities of luciferase and β-galactosidase were determined, as described previously 8.
