Bl005b

KCa3.1 inhibitor TRAM-34, NF-κB inhibitor PDTC and STAT3 inhibitor AG490 were purchased from MCE (Shanghai, China). All these inhibitors dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Antibodies against KCa3.1 (60276-1-Ig) and Interleukin (IL)-1β (16806-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies for inducible nitric oxide synthase (iNOS) (ab178945), matrix metalloproteinase (MMP)-9 (ab228402), vascular cell adhesion molecule (VCAM)-1 (ab134047), STAT3 (ab68153), phospho (p)-STAT3 (ab76315), horseradish peroxidase (HRP)-conjugated IgG (ab205718, ab205719) were purchased from Abcam (Cambridge, MA, USA). Antibodies against β-tubulin (15115S), NF-κB p65 (8242T), p-NF-κB p65 (3033T), p38 MAPK (8690T) and p-p38 MAPK (4511T) were purchased from CST (Danvers, MA, USA). Antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (BL006B) and β-actin (BL005B) were purchased from Biosharp (Hefei, China). Primary antibody for Histone H3 (EM30605) was purchased from HUABIO (Hangzhou, China)

Western blotting was performed as described in our previous study.29 (link) Briefly, the myocardium (with removal of atriums) was homogenized and lysed, and the extracted proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with 5% non-fat dry milk at room temperature for 2 hours, the blots were incubated overnight with primary antibodies against PTGS2 (Proteintech #12375-1-AP, China, 1:1000) and β-actin (Biosharp #BL005B, China, 1:2000) at 4°C. Subsequently, the membrane was incubated with the secondary antibody (Beyotime # A0208, China, 1:2000) for 2 hours at room temperature. The positive bands were visualized using the Ultra High Sensitivity ECL kit (Beyotime, China) and imaged using FluorChem FC3 (ProteinSimple, USA). The blots were densitometrically scanned using the ImageJ software (NIH, USA), and β-actin was selected as the internal reference according to previous studies.36 (link),37 (link)

Western blot assay was used to evaluate protein levels of AIM2 in sham-operated and injured spinal cords. The spinal cord segment (T8–10) was quickly dissected after intracardial perfusion of 200 mL physiological saline under anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneally; Beijing Dtftchem Technology Co., Ltd.). Spinal cord protein extraction and western blot assay were performed as described previously (Lin et al., 2018; Wang et al., 2018). The blots were incubated with primary antibody, rabbit polyclonal anti-β-actin (1:2000; BL005B; Biosharp, Hefei, China) or rabbit polyclonal anti-AIM2 (1:1000; 20590-1-AP; Proteintech Group, Rosemont, IL, USA), for 12 hours at 4°C. Afterwards, the blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit polyclonal secondary antibody (1:10,000; Cat# BL003A; Biosharp) for 1 hour at room temperature. Signals were detected with an enhanced chemiluminescence kit (Thermo Fisher, Waltham, MA, USA) following the manufacturer’s instructions. Finally, the signals were digitized, and densitometry was performed using ImageJ Software (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD, USA). AIM2 protein levels were normalized to β-actin (ratio of the gray values).