Psd95

Total protein in Tris-buffered saline (TBS) and neutralized formic acid (FA) extracts was quantified using the Ready to Use Bradford Reagent (Bio-Rad) and for TBS-Triton X100 (TBSX) extracts the BCA Protein Assay Kit (Pierce).
For western blot analysis of TBS-X fractions protein (20 μg) was separated on 4–12 % Bis-Tris gels (Invitrogen) and transferred onto low-fluorescence PVDF membranes. Membranes were blocked (1 hr. room temperature) with 5 % non-fat milk in TBS containing 0.1 % Tween 20 (TBST), then incubated (4 °C, overnight) with primary antibodies against post synaptic density protein (PSD-95, 1:1000, Cell Signaling), synaptophysin (1:1000, Cell Signaling), the EGF receptor (1:100, Santa Cruz Biotechnology) or actin (1 hr. room temperature, 1: 20,000, Cell Signaling) in TBST with 5 % bovine serum albumin (BSA). Membranes were incubated in secondary antibodies (Jackson Immunoresearch) with TBST wash (3X5min) in between steps and imaged using an Odyssey ® Fc Imaging System. Image J was utilized to quantify actin normalized proteins.

Protein was extracted from mice's hippocampal tissue. Protein lysates were separated by 12% SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 3% BSA for 1 h, the membranes were incubated with primary antibodies. CAMKII (Cell Signaling Technology, 7546, 1 : 500), P-CaMK-II (Cell Signaling Technology, 9546, 1 : 500), P-CREB (Cell Signaling Technology, 9198 s, 1 : 500), CREB (Cell Signaling Technology, 9197, 1 : 500), PKA (Proteintech, 55388-1-AP, 1 : 1000), NMDAR1 (Cell Signaling Technology, 5104 s, 1 : 1000), BDNF (Cell Signaling Technology, 47808, 1 : 1000), PSD95 (Cell Signaling Technology, #2507, 1 : 1000), GAPDH (Proteintech, 6004-1-lg, 1 : 2000) and Tubulin (Proteintech, 10094-1-AP, 1 : 2000) were used at 4°C overnight. The next day, blots were washed 4 times in TBST, followed by incubation with secondary antibodies for 1 h. After washing for 3 times, the blots were visualized using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). BDNF and pro-BDNF were normalized to Tubulin bands, and P-CERB and total CREB bands were taken as a ratio of Tubulin bands. All experiments were performed 3 times.

Hippocampal neuronal cells were rinsed with phosphate buffered saline (PBS), fixed in 4% paraformaldehyde for 8 min and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After being blocked with 5% milk for 30 min, the cells were incubated with primary antibodies conjugated to Alexa Fluor^®488 or 594 against microtubule-associated protein-2 (MAP2, Abcam, 1:250) and PSD-95 (Cell Signaling, 1:250) at 4 °C overnight. The secondary antibody was then incubated at room temperature for 1 h and rinsed in PBS three times. The nuclei were stained with DAPI (Sigma) for 5 min. Fluorescence images were obtained using a BX53 Olympus fluorescence microscope at 20 × magnification and captured using Olympus CellSens Standard software. Sholl analysis was applied to measure dendritic complexity. The length of dendritic arborization was analyzed and measured using a semi-automatized protocol via Imaris software (Bitplane, Inc.).

The two hippocampi were pooled from each freshly dissected mouse and the membrane fraction was isolated using the Mem Per Plus kit (Thermo Fisher Scientific, Waltham, MA). Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA). 20 μg protein was loaded on 4–12% Bis-TRIS gels and were transferred to a PVDF membrane. Proteins were detected using antibodies for 5-HT5A (LifeSpan Biosciences, Seattle, WA), β-Actin and PSD-95 (both from Cell Signaling).

tBHP, D-gal, AlCl₃, and glutamate were purchased from Sigma-Aldrich (St. Louis, MO). Kits used for MDA, SOD, glutathione (GSH), the bicinchoninic acid (BCA) protein assay, the ROS assay, and mitochondrial membrane potential detection were purchased from Beyotime (Shanghai, China). Antibodies against Nrf2, HO-1, SOD2, NQO-1, and Lamin B1 were obtained from Abcam (Cambridge, MA). Antibodies against β-actin, phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, GAPDH, SYN, PSD95, Bcl-2, and Bax were purchased from Cell Signaling Technology (Beverly, MA). All secondary antibodies (horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG) were purchased from Cell Signaling Technology (Beverly, MA). All reagents used were of the highest grade available commercially.