Chemiluminescent hrp substrate

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).

Cell proteins were mixed with SDS-PAGE Protein Loading Buffer and incubated at 95 °C for 5 min. Proteins were size-separated by SDS-PAGE (10% acrylamide gel, 130 V, 90 min), transferred onto a nitrocellulose membrane (220 mA, 120 min), blocked for 1 h with 5% BSA, and washed 3 times for 15 min with 0.2% Tris-Buffered Saline with Tween 20 (TBS-Tween). Membranes were incubated overnight with primary antibodies, washed 3 times for 15 min with 0.2% TBS-Tween, incubated for 1 h with HRP-conjugated secondary antibodies, and washed 3 times for 15 min with 0.2% TBS-Tween. After 5-min incubation with chemiluminescent HRP substrate (Thermo Scientific), bands were visualized by Chemidoc XRS System (Clinx Science Instruments, Shanghai, China) and analyzed by Image Lab Software (Clinx Science Instruments, Shanghai, China). The antibodies used were mouse anti-LPL (Santa, sc-73,646) (1:200 dilution), rabbit anti-GAPDH (Santa, sc-69,778) (1:2000 dilution), goat anti-rabbit IgG H&L (HRP) (Abcam, ab6721) (1:10000 dilution), and rabbit anti-mouse IgG H&L (HRP) (Abcam, ab6728) (1:5000 dilution).