Glur1

Brains of mice were removed followed by dissection of the hippocampus, which were homogenized using a dounce homogenizer. Tissue extracts were sonicated and centrifuged at 1000rpm. Supernatants were collected and a BCA assay was performed to measure protein levels. Bis-Tris polyacrylamide gels (4–12%) were loaded with 15–20ug of protein. Protein was transferred to PVDF membranes overnight. Membranes were then blocked in 5% milk or 5% BSA for 1hr at room temperature. After blocking, the membranes were incubated in primary antibody overnight at 4°C. Antibodies used: phospho-ERK1/2 (Cell Signaling, 1:3000), ERK1/2 (Cell Signaling, 1:3000), PSD95 (Thermo, 1:5000), GluR1 (Millipore, 1:1000), GluR2 (Millipore, 1:1000), NR2A (Millipore, 1:1000), NR2B (Millipore, 1:1000). Blots were washed and then incubated in HRP-conjugated secondary antibodies. The blots were then washed, treated with chemiluminescent substrate and imaged on the LI-COR Odyssey Fc. Densitometry analysis was performed using Odyssey imaging software and statistically analyzed using Graphpad Prism. Densitometry values for each sample were normalized to the average of the control animals and average densitometry of CKO animals were compared to control animals using t-tests.

Immunoblot samples of adult brain tissue and hippocampal slice cultures were sonicated in cold lysis buffer (Sigma-Aldrich; St. Louis, Missouri). Protein content was determined and equal amounts of protein were denatured in sample buffer and separated on gradient gels for subsequent transfer to nitrocellulose. Blots were incubated in blocking solution containing 5% milk or BSA for 1 h. Primary antibody staining utilized antibodies against cathepsin B (1:100, Calbiochem), GluR1 (1:1000; Millipore) and anti-αII spectrin (1:100, Santa Cruz), as well as against actin 20–33 (1:500, Sigma) and an antibody to gephyrin’s C-terminal (1:250) made against the sequence VELHKGEVVDVMVIGRL described in Kawasaki et al. (1997) (link). Anti-IgG-alkaline phosphatase conjugates and anti-IgG-horseradish peroxidase conjugates were used for the secondary antibody step, and antigen staining and image development involved the chemiluminescence protocols using the GE Amersham AI600RGB imager. Immunostained bands were scanned at high resolution to determine integrated optical density with BIOQUANT software (R & M Biometrics, Nashville, Tennessee).

PSD and synaptosomal fractions of the striatum, cortex, and cerebellum were prepared as previously described^{21 (link)}. Purified fractions were separated on SDS-PAGE and quantified using Odyssey Licor. β-Actin and Tubulin were used as loading controls. Specific primary antibody for SAPAP3 was prepared as previously described^{21 (link)}. Commercial antibodies used include SHANK3 (Santa Cruz SC-30193), GluR1 (Millipore MAB2263), GluR2 (Neuromab 75-002), NR1 (BD Biosciences 556308), NR2A (Millipore 07-632), NR2B (Millipore 05-920), Homer1 (Chemicon AB5877, Synaptic Systems 160022), Homer3 (Synaptic Systems 160303), mGLUR5 (Abcam ab76316), CaMKIIa (Millipore 05-532), Shank1 (Synaptic Systems 162002), Shank2 (Cell Signaling 12218S), B-Actin (Sigma A5441), and Tubulin (Sigma T5168). Statistical analysis was done using two-tailed Students’ t-tests.