Rabbit anti syngap

The extraction of total protein from cell culture of hippocampal neurons and immunoblot analysis were performed as previously described (Varela-Nallar et al., 2009 (link); Codocedo et al., 2012 (link)). The following primary antibodies were used: rabbit anti-pSynGAP (1:1.000; ABCAM), rabbit anti-SynGAP (1:1.000; ABCAM), mouse anti pCAMKII (1:1.000, Santa Cruz), and anti GAPDH (1:10.000, Santa Cruz). Primary antibodies were recognized using either a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:7000, Thermo Scientific) or an HRP-conjugated rabbit anti-mouse antibody (1:7.000 Thermo Scientific). The secondary antibodies were detected through enhanced chemiluminescence using the ECL Plus Western blotting detection system (GE Healthcare). Densitometric analysis was performed using NIH ImageJ software.

Protein concentration was determined by the BCA protein assay kit. Proteins (20 μg) were resolved in SDS-polyacrylamide gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Nonspecific binding sites on the blotting membrane were blocked with 5% non-fat dry milk for 2 h and incubated at 4°C overnight with mouse anti-NR2B (1∶1000, Abcam), rabbit anti-SAP102 (1∶500, Santa Cruz), rabbit anti-CaMKII (1∶1000, Cell Signaling), rabbit anti-SynGAP (1∶1000, Abcam), rabbit anti-pERK1/2 (1∶2000, Cell Signaling), rabbit anti-pCREB (1∶1000, Cell Signaling Technology) and rabbit anti-BDNF (1∶1000, Sigma). The proteins were detected with anti-rabbit or anti-mouse secondary antibody for 2 h at room temperature. Then, the proteins were visualized with BeyoECL Plus reagents and captured by an enhanced chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.).