Mouse anti gapdh

Western blot (WB) was performed according to a previous method (22 (link)). Briefly, after animals were anesthetized and decapitated, spinal cord tissues of the lesion site were removed and collected immediately on ice (8 (link)). Then, samples were homogenized and lysed in RIPA buffer containing protease and phosphatase inhibitors. After centrifuging at 12,000 rpm for 10 min at 4°C, protein concentrations were determined using a BCA Assay Kit (Beyotime). Equal amounts of protein lysate (20 μg) were separated by 10% SDS-PAGE electrophoresis, followed by transferring onto PVDF membranes. After blocking in 5% fresh-non-fat skim milk prepared in TBST for 2 h at room temperature, membranes were incubated with the appropriate primary antibodies at 4°C overnight. Then, membranes were incubated with corresponding HRP-conjugated secondary antibodies for 2 h at room temperature after washing with TBST. Finally, protein bands were visualized with chemiluminescent HRP Substrate (Thermo Fisher) under Western Lightning-ECL (Bio-Rad, USA). The following primary antibodies were used: mouse anti-Histone H3 (citrulline R2+ R8 +R17) (Abcam; 1:1000), rabbit anti-ZO-1 (Abcam, 1:5000), rabbit anti-occludin (Abcam, 1:5000), rabbit anti-transient receptor potential vanilloid type 4 (TRPV4) (Abcam; 1:1000), and mouse anti-GAPDH (Zen-bio, 1:5000).

Collected spinal cord tissues were lysed in radio immunoprecipitation assay lysis buffer (NCM Biotech) containing protease and phosphatase inhibitors. After ultrasonic homogenization, proteins were extracted by centrifugation, and the protein concentration was determined using a bicinchoninic acid assay. Following separation by 8% sodium dodecyl-sulfate polyacrylamide gel electrophoresis, protein samples were transferred to polyvinylidene difluoride membranes. The membranes were blocked in NcmBlot blocking buffer (NCM Biotech) for 15 min and subsequently incubated with primary antibodies as follows (4°C, overnight): rabbit anti-STAT2 (1:500, HuaBio), mouse anti-GAPDH (1:5000, ZENBIO), and mouse anti-H3 (1:1000, Cell Signaling Technology). The polyvinylidene difluoride membranes were washed with TBST buffer, followed by incubation with the corresponding secondary mouse or rabbit antibody (1:5000, goat anti-mouse, goat anti-rabbit, ZENBIO) for 2 h at room temperature. The membranes were developed using a ChemiDoc XRS System and Image Lab software (Bio-Rad, Universal Hood III, United States).