Mouse anti histone h3

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).

Proteins were sonicated, boiled in sample buffer (1% SDS, 16.7 mM Tris–HCl pH 6.8, 5% sucrose, 0.67 mM EDTA, 10% B-Mercaptoethanol (v/v)) and resolved using SDS-PAGE [78 (link)] with 10–15% polyacrylamide gels and 8 M urea in the stacking gel for better resolution of chromosomal proteins (electrophoresis apparatus; BioRad). For gel staining, protein bands were visualized using InstantBlue (Expedeon). For immunoblotting, proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in PBS-0.1% Tween for 1–2 h.
Primary antibodies used for immunoblotting included rabbit anti-GgCENP-T (1 : 2000) [6 (link)], rabbit anti-GgNdc80 (1 : 2000) [43 (link)], mouse anti-GgINCENP (1 : 3) [79 (link)], mouse anti-Histone H3 (1 : 500; Abcam), rabbit anti-GgTopo IIalpha (1 : 500) [80 (link)], mouse anti-alpha Tubulin (1 : 2000; B512 Sigma) and anti-GgCyclin B2 [81 (link)]. When using the LI-COR Odyssey system, membranes probed with secondary antibodies (IRDye 800 CW/IRDye 680; LI-COR Biosciences) were washed for at least 45 min with 0.1% Tween in PBS and a final wash performed with PBS for 5 min. Median fluorescence intensities for individual protein bands were subsequently determined using a CCD scanner (Odyssey; LI-COR Biosciences).

Western blot analysis was performed as previously described^{43 (link)} using the following antibodies: mouse anti-NOXA, rat anti-BMF, rabbit anti-MCL-1 (Enzo Life Science, Farmingdale, NY, USA), mouse anti-BCL-2, rabbit anti-BAK (BD Biosciences), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-BIM, mouse anti-PARP, rabbit-anti PUMA, rabbit-anti BCL-x_L (Cell Signaling, Beverly, MA, USA), mouse anti-GAPDH (HyTest, Turku, Finland), rabbit anti-H3K4me2 (Diagenode, Liège, Belgium), rabbit anti-acetylated histone H3 (Merck Millipore, Darmstadt, Germany) and mouse anti-histone H3 (Abcam) or mouse anti-β-Actin (Sigma, Germany). Goat anti-mouse, goat anti-rabbit and goat anti-rat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odysee Imaging System, LI-COR Biosciences, Bad Homburg, Germany) were used for detection. For detection of histone modifications cells were lysed using RIPA buffer supplemented with Pierce Nuclease (Thermo Fisher, Waltham, MA, USA). Representative blots of at least two independent experiments are shown.