Proteins denatured by heating for 15 min at 60°C in Laemmli sample buffer (Laemmli, 1970) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking in TTBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, and 5% skim milk powder), the membranes were incubated with primary antibody overnight at 4°C and then with horseradish peroxidase (HRP)-secondary

antibody for 1 hr at room temperature. Detection was performed using an enhanced chemiluminescence (ECL) kit and Hyperfilm http://www.selleckchem.com/products/epacadostat-incb024360.html MP, and quantified using a Fuji BAS-2000 image analyzer. Hippocampal neurons (DIV14–21) were incubated with neurobasal medium for 5 min (control) or with neurobasal medium containing 100 μM NMDA for 5 min at 37°C, washed twice with neurobasal medium and then maintained in the medium for 15 min or time indicated in results. Additional information on materials, antibodies, DNA constructs, protein purification, slice in Situ hybridization, FISH in cultured hippocampal neurons, tracking mRNA using MS2 system, live imaging using Dendra-Kv4.2, immunoprecipitation of FMRP in brain lysates, quantitative RT-PCR, biotinylated RNA and protein binding assay, immunohistochemistry, Selleck C646 surface biotinylation, surface staining, in vitro translation assay, luciferase reporter assay and LTP experiment are described in Supplemental

Experimental Procedures. We thank Drs. Tom Schwarz and Jeanne M. Nerbonne for kindly providing Kv4.2 KO mice, Dr. Lynn Regan for kindly providing mouse full-length FMRP construct, Dr. Stephanie Ceman for kindly providing GFP-FMRP constructs, Dr. Marc I. Diamond for kindly providing pcDNA3-mouse H1d construct, Dr. Seung Key Jang for sharing MS2-GFP-NLS and MS2BS(6X) constructs, and Dr. Philippe Mourrain and Gemini Skariah for the

help and support they provided for the in situ hybridization procedure. We also thank Denan Wang for maintaining mouse lines, Marena Tynan-La Fontaine for genotyping, Dr. Sila Konur for help with imaging and for critical reading of the manuscript and for Dr. Desiree Thayer for critical reading of the manuscript and many helpful discussions. This work was supported by the National Institute of Mental Health (L.Y.J.). Y.N.J. and L.Y.J. are investigators of the Howard Hughes Medical Institute. “
“Place cells in the hippocampus encode information about an animal’s location in its environment by means of spatially restricted firing patterns called place fields (O’Keefe and Dostrovsky, 1971 and O’Keefe and Nadel, 1978). Recent findings have demonstrated that spatial information to the hippocampus is provided by layer II and layer III entorhinal cortex (EC) neurons—grid cells—which fire at regular, triangularly spaced locations as an animal traverses its environment (Hafting et al., 2005).