Protease inhibitor solution

The cells were lysed with lysis buffer (Beyotime, Shanghai China) supplemented with a protease inhibitor solution (Beyotime) and centrifuged at 12 000 g for 10 min at 4 °C. Extracted proteins were separated on 10–15% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 37 °C, the membranes were incubated overnight with primary antibodies at 4 °C, washed with TBST, and then incubated with secondary antibodies. The western blot results were quantified by densitometric analysis using the Quantity One 4.6.9 software (Bio-Rad).
The following antibodies were used: anti-cleaved caspase-9 (9509T, CST), anti-cleaved caspase-3 (9664T, CST), anti-Bax (#2772, CST), anti-Bcl2 (#3498, CST), anti-cytochrome c (10093, Proteintech), VDAC rabbit mAb (4661, CST), anti-MFN1 (NBP1-71775, Novus Biologicals), DRP1 rabbit mAb (8570, CST), OPA1 rabbit mAb (80471, CST), anti-FIS1 (D122377-0025, BBI life sciences), anti-MFN2 (12186, Proteintech), anti-beta tubulin (10094, Proteintech), anti-PSD95 (20665, Proteintech), spinophilin rabbit mAb (14136, CST, Boston), HP-goat anti-mouse (ZB-2305, Zsbio, Beijing, China), HP-goat anti-rabbit (ZB-2301, Zsbio, Beijing, China), and Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (33112ES60, Yeasen).

The animals were euthanized with an excessive dose of pentobarbital sodium. Both sides of the nodose ganglia and NTS were removed immediately and frozen on liquid nitrogen. Nine pairs of tissues from NG and 4 tissue samples from NTS were collected for Western blots. The tissues were lysed by using lysis buffer (Beyotime Biotech, China) containing 1% protease inhibitor solution (Beyotime). The lysate was centrifuged for 15 min to collect supernatant. The protein concentration was determined using BCA Protein Assay Kit (Beyotime). The samples were boiled for 7 min and then loaded on 10% SDS-PAGE gel (100 mg of protein, 15 ml per well) for electrophoresis with 110 V for 90 min. The protein on the gel was transferred onto NC membranes at 300 mA for 75 min, which was blocked by 5% nonfat dry milk diluted by PBS at room temperature for 3 hours. The membrane was probed with primary antibody (GluR2 receptor rabbit polyclonal antibody, Alomone Labs, 1:200) at 4°C overnight and then the secondary antibody (goat anti-rabbit, Licor, 1:8000) for 60 min at room temperature. GAPDH was used as the internal control. Bound bands were visualized and analyzed using Odyssey Infrared Imaging System (LI-COR Biosciences) and Odyssey v1.2 software.

All procedures for purified mitochondrial isolation were conducted at 4 °C. Briefly, cells and freshly prepared tissue were washed in 0.9(w/v) sodium chloride solution. The cells or tissue was homogenized with lysis buffer (Beyotime, Shanghai China) supplemented with a protease inhibitor solution (Beyotime) and then centrifuged at 1000 g for 10 min to remove nuclear contaminants, cell debris, and intact cells. The supernatant was transferred to a clean 1.5‐mL tube and centrifuged again at 6000 g for 10 min at 4 °C. The supernatant constituting the microsomal fraction was removed.