Rabbit anti gapdh

The exosomal and cellular proteins were extracted by DNA/RNA/protein Isolation Kit (catalog number: R6734-02, OMEGA). The sample was denatured by heating, separated by SDS-PAGE and transferred to a PVDF (polyvinylidene fluoride) membrane. Next, the membranes were blocked with 5% skimmed milk powder and separately probed with rabbit anti TSG101 (catalog number: GB11618, servicebio), rabbit anti HSP70 (catalog number: GB11241, servicebio), rabbit anti TSC1 (catalog number: GB11882, servicebio), rabbit anti calnexin (catalog number: 10427–2-AP, Proteintech), rabbit anti GAPDH (catalog number: GB11002, servicebio), rabbit anti β-actin (catalog number: GB11001, servicebio) and mouse anti DKK3(catalog number: 66758–1-Ig, Proteintech) overnight at 4 °C with a final dilution 1:1000 (v/v) in 5% milk. After three times of washing, the membranes were incubated with goat anti-rabbit secondary antibodies (GB23303, Sevicebio, Wuhan, China) or goat anti-mouse secondary antibodies (GB23301, Sevicebio, Wuhan, China) with 1:2000 dilution (v/v) at 37 °C for 1.5 h. The images of membranes treated with ECL (enhance chemiluminescence) were captured by Western Blotting Detection System (Tiangen, Beijing, China).

Proteins extracted from spinal cord tissues and primary microglia were separated by SDS‐PAGE and transferred onto nitrocellulose filters membranes. The membranes were blocked for 1 h at room temperature using 5% nonfat milk, and incubated overnight at 4°C with primary antibodies (1:1000). The following primary antibodies were used: rabbit anti‐STAT3 and anti‐phospho‐STAT3 (Cell Signaling), mouse anti‐IL‐6 and rabbit anti‐TNF‐α (Santa Cruz), rabbit anti‐β‐ACTIN and rabbit anti‐GAPDH (Servicebio). Next, the membranes were incubated with secondary antibodies for 1 h at room temperature (1:1000; Servicebio). Protein expression levels were normalized to β‐ACTIN or GAPDH internal controls.

Honokiol (HY-N0003), MK-801 (HY-15084B) and Compound C (HY-13418A) were purchased from MCE (Shanghai, China). BAPTA-AM was purchased from Thermo Fisher Scientific (Cat# B6769); cantharidin was purchased from Institute for Drug Control (Shanghai, China). All primary antibodies used in this study included: Rabbit anti-NMDAR2B (Abcam, Cambridge, UK); Fluorescein isothiocyanate (FITC) anti-SIRT3 (Biorbyt, Cambridge, UK); Rabbit anti-PGC-1α (Santa Cruz, CA, USA); Rabbit anti-AMPKα and Rabbit anti-phospho-AMPKα (Thr172) (Cell Signaling Technology, MA, USA); Rabbit anti-MAP2 (Abcam, Cambridge, UK); Mouse anti-NeuN (Abcam, Cambridge, UK); Rabbit anti- CaMKKβ and Rabbit anti-Phospho-CaMKKβ (Abcam, Cambridge, UK); Phosphatase 4 (Santa Cruz, CA, USA); Rabbit anti-GAPDH and Rabbit anti—beta Actin (Servicebio, WuHan, China).

Rat tissues in the medulla were freshly dissolved in RIPA buffer, which consisted of a protease inhibitor cocktail and phenyl‐methylsulfonyl fluoride (PMSF). In a 12.5% SDS‐PAGE gel, equal amounts of proteins (20 μg) were separated and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat dry milk, the membranes were incubated with mouse anti‐c‐Fos (1:2000, Proteintech) and rabbit anti‐GAPDH (1:3000, Servicebio) at 4°C overnight. The following morning, the membranes were washed using TBST and then incubated with HRP‐labeled goat anti‐mouse and HRP‐labeled goat anti‐rabbit (both 1:5000, Servicebio) for 1 h. The chemiluminescence system (ChemiDocTM XRS+, BioRad) was used to visualize protein blotting. Protein expression was quantified using Image J software 1.8.0.