Pglua1 s845

Synaptosomal fractions from DIV14 primary cortical neurons were prepared as described previously [60] (link),[61] (link). Surface biotinylation was performed according to the previous study [60] (link). Equal amounts of protein were loaded on 10% SDS-PAGE gel and transferred to the nitrocellulose membrane. Membranes were blotted with GluA1 (Millipore, 1∶5,000), GluA2/3 (Millipore, 1∶500), pGluA1(S845) (Millipore, 1∶1,000), calcineurin (Millipore, 1∶1,000 or Santa Cruz Biotechnology, 1∶500), actin (Sigma, 1∶5,000), pCREB (Cell Signaling, 1∶500), and CREB (Cell Signaling, 1∶1,000) antibodies and developed with ECL (Perkin Elmer). Synaptosomes were isolated from at least three independent cultures, and immunoblots were least duplicated for quantitative analysis.

Synaptosomal fractions from DIV14 primary cortical neurons were prepared as described previously [43 (link),46 (link),47 (link)]. Surface biotinylation was performed according to the previous studies [43 (link),46 (link),47 (link)]. For IP3R phosphorylation, whole cell lysates were collected as described previously [43 (link)]. Equal amounts of protein were loaded on 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blotted with GluA1 (Millipore, 1:2,000), GluA2 (Abcam, 1:2,000), pGluA1(S845) (Millipore, 1:1,000), pIP3R(S1756) (Cell signaling, 1:1,000), tubulin (Sigma, 1:5,000), PSD95 (Neuromab, 1:2,000), synaptophysin (Sigma, 1:5,000), NR1 (Millipore, 1:1,000), and actin (Abcam, 1:2,000) antibodies and developed with ECL (Thermo Fisher Scientific). Synaptosomes were isolated from at least three independent cultures, and immunoblots were at least duplicated for quantitative analysis.