Myc-NGL-2 and myc-NGL2∗ were both subcloned into the pEF-BOS

(Mizushima and Nagata, 1990) vector downstream of the elongation factor promoter. shNGL-2 was subcloned into the pSUPER/Neo vector (Oligoengine) downstream of the H1 promoter. The H1 promoter and shNGL-2 were then subloned into the PacI site of FCK(0.4)GW (a gift from Dr. Pavel Osten, Cold Spring Harbor Laboratory) lentiviral backbone upstream of the CamKII promoter, which contains a 0.4 kb fragment of mouse CamKII protomoter-driving MDV3100 mouse EGFP (Dittgen et al., 2004). FCK(0.4)GW was used as a control. NGL-2 deletion constructs were as follows: NGL2∗ΔLRR (aa 79–287 deleted from full-length mouse NGL2∗) and NGL2∗ΔPDZ (aa 1–648 of full-length mouse NGL2∗). NGL-2-GFP fusion was generated Selleck OSI744 by sequentially subcloning NGL-2 cDNA obtained from

Open biosystems (Thermo Fisher Scientific) in frame with GFP into the pEF-BOS vector downstream of the elongation factor promoter. All constructs were sequenced to verify integrity. NGL1(NGL2LRR) (originally termed pCA NGL1r123-mVenus) was a gift from Elena Seiradake and Alexandru Radu Aricescu. In situ hybridizations were performed as described (Pasterkamp et al., 1999), using 20 μm horizontal P7 and P14 rat brain cryosections. P28 mice were deeply anesthetized with isofluorane, decapitated, and brains were harvested, flash frozen, and stored at −80°C. Crude membranes were isolated by homogenizing each brain in 5 mL homogenization buffer (0.32 mM sucrose, 4 mM HEPES [pH 7.5], and protease inhibitors) using a Dounce homogenizer. Homogenate was spun at 3,000 rpm for 10 min at 4°C. Supernatant (S1) was collected and spun at 10,000 × g for 15 min at 4°C. Each pellet (P2) was resuspended in homogenization buffer and spun at 10,000 × g for 15 min at 4°C. Pellets (P2′) were lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1% Triton-X, 0.5 M EDTA, protease inhibitors) and rocked for 30 min at 4°C. Samples were centrifuged at 10,000 × g for 20 min at 4°C, and supernatant was removed and mixed with sample buffer

for analysis by western blot. Western blots were probed with mouse anti-NGL2 (Clone N50/35, NeuroMab) and Adenosine rabbit anti-βIII tubulin (Abcam). P14 WT and KO littermate mice were given a lethal dose of sodium pentobarbital and perfused with PBS, followed by 4% paraformalydehyde (PFA) in PBS. We cut 100 μm coronal sections with a vibrating microtome (Vibratome), then blocked them in PBS containing 3% bovine serum albumin and 0.2% Triton X-100 (Sigma) for 1 hr at room temperature, and then immunostained them using standard procedures. See Supplemental Experimental Procedures for more information. P7 mice were given a lethal dose of sodium pentobarbital and perfused with PBS, followed by 4% PFA in PBS. DiI crystals (Invitrogen) were placed in CA3 or EC of fixed brains.