Psd95

Cultured cells and brain tissues (n = 6 per group) from the cortex and hippocampus were homogenized at 4°C in radioimmunoprecipitation assay buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM EGTA, pH 8.0, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and 1% Triton X-100] with protease inhibitor (P8340; Sigma Aldrich). The homogenate was centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was diluted in SDS sample buffer (62.6 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.01% bromophenol blue) and sonicated for 10 s after incubation at 100°C for 5 min. The lysates were subjected to 10% SDS–polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membrane. After blocking non-specific sites with Tris-buffered saline (TBS) containing 5% defatted dried milk, membranes were probed with the antibodies. Anti-β-actin mouse antibody (Affinity) blotting was used as a loading control. Rabbit antibodies were CHIP (proteintech), synapsin I (SYN I, proteintech), phosphorylated synapsin I S9 (SYN I-S9, Affinity), S427 (SYN I-S427, Affinity), S605 (SYN I-S605, Affinity), ubiquitin (proteintech), SNAP25 (Affinity), and PSD95 (Affinity).

Total protein was extracted from peri-infarcted brain tissues, and total protein concentration was determined with a BCA kit (Solarbio, China). Proteins were separated by polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% non-fat milk for 1 h at room temperature and with anti-Syn (1:2,000; Abcam, United Kingdom), PSD-95 (1:1,000; Affinity, China), and β-tubulin (1:3,000; Solarbio, China) antibodies at 4°C overnight. Then, the cells were incubated with the IgG antibody (1:1,000; Cell Signaling Technology, Danvers, MA, United States) for 1 h at room temperature. Visualization was performed on a gel electrophoresis imager using an ECL hypersensitive chemiluminescent solution (Millipore, Germany).