17β estradiol e2 ici 182 780

17β-Estradiol (E₂), ICI 182,780 (ICI; an ER antagonist), tamoxifen, clomiphene citrate, U18666A, and filipin were purchased from Sigma-Aldrich (Sigma, US). D-erythro-sphingosine (Sph) and C17-d-erythro-sphingosine (C17-Sph) were purchased from Avanti Polar Lipids (Alabastar, US). High-affinity (with the strong calcium buffering) Rhod-dextran was purchased from Thermo Fisher (Life technology, US). DMSO (Sigma, US) was used as solvent for tamoxifen and clomiphene citrate, PBS was used for U18666a and high-affinity Rhod-dextran, and methanol (Sigma, US) was used for Sph and C17-Sph.

17β-Estradiol (E₂), ICI182,780 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were obtained from Sigma (St. Louis, MO, USA);Dulbecco modified eagle medium (DMEM)and other cell culture supplements were purchased from GIBCO (Grand Island, NY, USA); Dimethyl sulfoxide (DMSO) was from Sinopharm Chemical Reagent Co., Ltd.(Beijing, China). Charcoal-dextranfetal bovine serum (CD-FBS) was bought from biological industries (Kibbutz Beit-Haemek, Israel); Estrogen receptor α antibody, estrogen receptor β antibody, Ki-67 antibody and pS2 antibody were purchased from Abcam (Cambridge, UK). LanthaScreen TR-FRET ERα and ERβ competitive binding assay kits were obtained from Life Technologies (Carlsbad, California, USA); The dual-luciferasereporter assay system were provided from Promega (Madison, USA). All other chemicals solvents used for high-performance liquid chromatography (HPLC) were of HPLC grade.

17β-estradiol (E2), ICI 182,780 and DMSO were purchased from Sigma (St. Louis, MO). Triton X, protease inhibitors and LightCycler® 480 SYBR Green I Master were purchased from Roche Molecular Biochemicals (Indianapolis, IN). GPER-1 C-terminal antibody was kindly provided by Dr. Eric Prossnitz, GPER-1 N-terminal antibody was purchased from Abcam, (Cambridge, MA), and Zenon Alexa Fluor Labeling Kit from Molecular Probes, (Eugene, OR). G-1 was purchased from Calbiochem (San Diego, CA) and ³H-testosterone from PerkinElmer Life Science (Boston, MA). MTS Titer Cell Proliferation assay was purchased from Promega Corporation (Madison, WI). Ovine Luteinizing Hormone was provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). Except for estradiol, which was diluted in 30% ethanol:70% Dimethylsulfoxide (DMSO), all ligands were solubilized in DMSO and kept as 1 mM stock solutions at −20°C.