3h myo inositol

Sulfo-NHS-SS-biotin and Neutravidin were purchased from Thermo Scientific (Waltham, MA, USA. Mouse anti-FMRP antibody was purchased from Abcam (Cambridge, UK). DHPG was purchased from Tocris Bioscience (Bristol, UK). Myo-[³H]-inositol was purchased from Perkin Elmer (Waltham, MA, USA). Dowex I-X8 resin, rabbit anti APP antibody, rabbit anti-phospho mTOR (pSer2481) antibody and rabbit anti-mTOR antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA).Goat anti-rabbit secondary antibody was purchased from BioRad (Hercules, CA, USA). Aggregated Aβ human ELISA kit and rabbit Aβ peptide antibody were purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-mGluR5 was purchased from Millipore (Billerica, MA, USA). Finally Vector Elite ABC kit rabbit and mouse, and Vector SG substrate was purchased from Vector laboratories. All other biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Ligand-induced activation of the human D_2L receptors was studied employing inositol phosphate (IP) accumulation assays as described54 (link)55 (link). Briefly, HEK 293 cells were transiently co-transfected with cDNA encoding for D_2L and the hybrid G protein Gα_qi5 (Gα_q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gαi; gift from the J. David Gladstone Institutes). Twenty-four hours after transfection, cells were transferred into 24-well plates. After adding myo-[³H]inositol (specific activity = 22.5 Ci mmol⁻¹, PerkinElmer) and incubation for 15 h, medium was aspirated, the cells were washed with serum-free medium supplemented with 10 mM LiCl, and test compounds 37 °C. Then, cells were lysed by adding 0.1 M NaOH. After neutralization with formic acid, the cell extract was separated by anion-exchange chromatography using an AG1-X8 resin (Bio-Rad) by washing and finally eluting total IP directly into scintillation counting vials. Radioactivity was determined by scintillation counting in a Beckman LS 6500 (Beckman). Data were analyzed by normalizing disintegrations per minute (d.p.m.) values; this was done by setting the data for non-stimulated receptor (buffer) equal to zero and the effect for quinpirole equal to 100%.

96-well plates were coated with poly-D-lysine and HEK-293 cells were plated (35.000 cells/well). Cells were transfected for 5 h the following day and subsequently incubated O/N with 0.5 μCi/mL myo[3H]inositol (Perkin Elmer) in 100 μL growth medium. On day 3 cells were washed with 200 µL HBSS/well (Gibco, Life Technologies) followed by a pre-incubation (30 min, 37 °C) with 100 µL HBSS supplemented with 10 mM LiCl. Cells were stimulated with ligand (120 min, 37 °C) and lysed with 50 µL 10 mM formic acid (30 min on ice). In a white 96 well plate 20 µL cell extracts and 80 µL 1:8 diluted YSi scintillation beads (Perkin Elmer) were mixed. The plate was spun down, and a Packard Top Count NXT counter recorded light emission (scintillation) after an 8 h delay.