Goat anti rabbit or anti mouse secondary antibodies

Cellular protein was extracted using RIPA buffer (Beyotime) containing phenylmethylsulfonyl fluoride and phosphatase inhibitors. Equal amounts of extracted cellular protein were resolved on 6–12% SDS-PAGE-denaturing gels and transferred electrophoretically onto polyvinylidene difluoride membranes. The membranes were blocked with 5% no-fat milk for 2 h at room temperature. The bands were incubated with the following diluted primary antibodies: p-AMPK, AMPK, p-mTOR, mTOR, Beclin-1, LC3B, γH2A.X (Cell Signaling, MA, USA, 1 : 1000), P21, P16 (Abcam, Cambridge, UK, 1 : 1000), Rad51 (Abcam, UK, 1 : 5000), and β-actin (Boster, Wuhan, China, 1 : 5000) overnight at 4°C. After washing three times with TBST, the membranes were then incubated for 2 h at room temperature with goat anti-rabbit or anti-mouse secondary antibodies (Boster, Wuhan, China, 1 : 5000). The excessive secondary antibody was washed off three times with TBST, and the bands were visualized using the Bio-Rad imaging system with the ECL imaging kit (Millipore Corporation). The intensity of the images was quantified by using ImageJ software.

On day 9, at 12 hours after administering NTG/VEH, the mice were deeply anesthetized with 1% pentobarbital sodium, and the brain TNC tissues of the medulla oblongata were quickly harvested on ice, frozen in liquid nitrogen, and stored at − 80 °C. The TNC tissues were sonicated in RIPA lysis buffer, added with 1 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF, 20 mM NaF, and 2 mM protease inhibitor cocktail, and quantified using the Bradford method. Proteins were separated using 8%–12% SDS-PAGE gels and were transferred to the PVDF membrane (Millipore). The PVDF membranes were blocked with 5% milk for 2 hours at room temperature, and were incubated overnight at 4 °C with the following primary antibodies: anti-c-Fos (1:1000; ab222699 Abcam), anti-CGRP (1:200; sc-57,053 Santa Cruz), anti-VGluT1 antibody (1:1000; ab227805 Abcam) and anti-GAPDH (1:5000; YM3445 ImmunoWay). Then, the membranes were washed in TBST and incubated with goat anti-rabbit or anti-mouse secondary antibodies (1:1000; Boster) for 1 hour at room temperature. The bands were visualized with Chemiluminescent HRP substrate (Millipore) on Azure Biosystem C500 and quantified using ImageJ.