C A schematic diagram highlighting possible proteinCprotein interaction motifs shared between GluNR2A and GluNR2D Refinement of the CtBP1 binding site on GluN2A was determined using a series of GluN2A C-terminal deletion constructs. fish constructs. Mammalian Cell Transfections Human embryonic kidney (HEK) 293 CIP1 cells were cultured and transfected using the calcium phosphate method as previously described . Cells were incubated post-transfection in the presence of 1?mM ketamine to prevent NMDA receptor-mediated cytotoxicity . HEK 293 cells were transfected in parallel with either CtBP1FLAG alone (10?g) or, GluN1-C2?+?GluN2A?+?either CtBP1, PSD-95 or SAP102 clones. The ratio of DNAs for the transfections were GluN1: GluN2: CtBP1, PSD-95 or SAP102, 1:3:4 which corresponded to 2.5?g:7.5?g:10?g for a 20?g total DNA for transfection of a 1??250?ml flask. For transfections where cell surface NMDA receptor expression was measured, HEK 293 cells were subcultured overnight prior to transfection in poly-d-lysine (100?g/ml)-coated 24-well dishes and 0.5?g total plasmid DNA was used per well. Immunoprecipitation Assays HEK 293 JNJ-10397049 cells were harvested 24?h post-transfection, cell homogenates prepared and solubilised for 1?h at 4?C at a concentration of 1 1.5?mg protein/ml with solubilisation buffer (50?mM Tris-citrate, pH 7.4, 240?mM NaCl, 5?mM EDTA, 5?mM EGTA, 1% (v/v) Triton X-100 containing benzamidine (1?g/ml), bacitracin (1?g/ml), soybean trypsin inhibitor (1?g/ml), chicken egg trypsin inhibitor (1?g/ml) and phenylmethylsulphonyl fluoride (1?mM)). Samples were diluted to 1 1?mg protein/ml with the above solubilisation buffer and solubilised material was collected by centrifugation at 100,000g for 40?min at 4?C. The detergent extracts were incubated with affinity-purified rabbit anti-GluN1 C2 antibodies (5?g) or protein A purified non-immune rabbit Ig (5?g) as control overnight at 4?C. Protein A Sepharose (25?l) was added and samples incubated for 1?h at 4?C. Immune pellets were collected by centrifugation for 15?s at 600g, washed with 3??1?ml solubilisation JNJ-10397049 buffer and then analyzed by immunoblotting. For immunoprecipitations from native tissue, the P2 membrane fraction was made from adult rat brain minus the cerebellum and 1% (w/v) sodium deoxycholate detergent solubilised 100,000g extracts were prepared as described . Samples were incubated with anti-GluN1 C2 or non-immune rabbit Ig primary antibodies for 16?h at 4?C. Immunoprecipitations were completed as above. Immunoblottting Immunoblotting was performed as previously described using 25C50? g protein/sample precipitated using the chloroform/methanol method and SDS-PAGE under reducing conditions in 7.5% (w/v) polyacrylamide slab minigels all as previously described . Primary antibodies used were: anti-GluN1 C2 (911C920), anti-GluN2A JNJ-10397049 (1381C1394) or anti-FLAG M2 primary antibodies. Rabbit or mouse horseradish-linked secondary antibodies (GE Healthcare, Chalfont St Giles, Bucks., UK) were used at a final dilution of 1 1:2000 and immunoreactivities were detected using the ECL western blotting system. Determination of NMDA Receptor Cell Surface Expression by ELISA NMDA receptor cell surface expression was carried out by ELISA assay using affinity-purified antibodies directed against an extracellular epitope of GluN2A i.e. anti-GluNR2A 44C58 Cys at a concentration of 0.125?g/ml all exactly as described in . Results and Discussion cDNA Library Screening: Identification of a Putative NMDA GluN2D Interacting Protein The screen of the mouse brain cDNA library with the bait, pGBKT7GluN2D 864C1323, yielded 240 positive interactors. From these, 60 strong and 60 weak interactors were randomly selected and each were subjected to yeast mating assays. From the selected 120 clones, 20 (19 from the pool of strong interactors and 1 from the pool of weak interactors) were real interactors in the yeast mating assays. For these 20 clones, each colony was analysed in triplicate to determine the presence of a cDNA insert, the size of the insert and if the insert was in frame with the activation domain. Ten clones satisfied these criteria. These were subsequently DNA sequenced. Within these ten, six clones were eliminated as they are.