Kz 3 fp

Trigeminal ganglion from each mouse was homogenized with a tissue grinder (Servicebio, KZ-III-FP, China), and total protein was extracted using RIPA buffer (Beyotime, P0013B, China) according to the manufacturer protocol, containing 1% phenylmethylsulfonyl fluoride (Beyotime, ST506, China). The protein concentration was measured by BCA protein assay kit (Beyotime, P0010S, China). Equal amounts of protein were then subjected to electrophoresis on 12% SurePAGE, Bis-Tris gels (Genscript, M00669, USA) followed by electrophoresis transfer to PVDF membranes (Millipore, IPVH00010, USA). Membrane blocking was performed with 5% skim milk (BD, 232100, USA) at room temperature for 2 h. Primary antibodies were used rabbit anti-GRP78 (1:1,000, Abcam, ab21685, USA), and anti-GAPDH (1:20,000, Proteintech, 10494-1-AP, China). Secondary antibody used was goat anti-rabbit IgG (1:10,000, Cell Signaling Technology, 7074, USA). The Omni-ECL femto light chemiluminescence kit (EpiZyme, SQ201, China) was used to visualize the bands. Immunoreactivity was detected using automatic chemiluminescence (Tanon, 5200, China). Image J2 was used to semi-quantify the blot images.

The proteins of the spinal cord (epicenter ± 5mm) were extracted by protein extraction buffer (Beyotime, P00103J) supplemented with 1% protease inhibitor (Beyotime, P1005) in a tissue grinding machine (Servicebio, KZ-III-FP). Cell lysates were centrifuged at 12000g for 15min at 4°C to collect supernatants. The protein concentration was determined by the BCA kit (Beyotime, P0012) and followed by denaturing at 95°C for 10 min in 1×SDS loading buffer. Subsequently, samples with an equal amount of protein were loaded and applied to 10% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore). The membranes were sealed and then incubated with specific primary and secondary antibodies. Proteins were visualized using enhanced chemiluminescence substrate (Tanon) and then quantified using a Tanon Chemiluminescent Imaging System.