Ecl chemiluminescence system

We prepared protein extracts from rats' brain tissue and HT22 cells. For rat's brain tissue, Briefly, rats were killed by decapitation; whole brains were removed and frozen at −80°C brain. Tissue lysates were prepared from frozen brain tissues, brain tissues were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche). Next, protein concentrations in the supernatant were determined using a BCA assay kit (Beyotime, Shanghai, China). An equal volume of 15 μg of proteins (extracted from the rats brain tissue and HT22 cells) were separated on 5–10% SDS-PAGE gels and transferred to the polyvinylidene difluoride membrane followed by blocking in 5% skim milk, next, the following antibodies were added to detect the primary antibodies: PDE10A (1 : 1000), TNF-α (1 : 1000), IL-1β (1 : 1000), IL-6 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1500), caspase-3 (1 : 1000), NLRP3 (1 : 1000), caspase-1 (1 : 1500), and GAPDH (1 : 2000). Next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 3000) for 1 h; the immunoblots were developed using an ECL chemiluminescence system, according to the manufacturer's instructions (Beyotime, China), and analyzed with Image J software (NIH, Bethesda, MD, USA).

Western blot analysis was performed to assess protein levels of apoptosis and autophagy-related proteins in the brains of RNS and control, using our standard methods as described previously (Gao et al., 2017 (link)). In brief, proteins were extracted from brain tissue and equal amounts of protein were separated by gel electrophoresis and transferred onto Hybond-polyvinylidenedifluoride (PVDF) membranes. After incubating with primary antibodies to anti-IL-33 (1:500, R&D), anti-NF-κB (1:500, CST), anti-ST2L (1:500, abcam), anti-LC3B (1:3000, abcam), anti-Beclin-1 (1:1000, bioworld), anti-P62 (1:1000, abcam), anti-cleaved-caspase-3 (1:500, bioworld), anti-Bcl-2 (1:500, abcam) and anti-β-actin (1:10,000, Sigma). anti-β-actin was used as a loading control. Then, the PVDFs were incubated with the respective HRP-conjugated secondary antibody for 2 h at room temperature. Blots were detected with the ECL chemiluminescence system (Beyotime Institute of Biotechnology) and were captured on autoradio graphic films (Kodak). Films were scanned and densitometric analysis of the bands was performed with Sigma Scan Pro 5.

Protein levels of apoptotic and autophagy-related proteins in brains of TBI and Sham groups were detected by western blot analysis, using the standard methods we described previously (Luo et al., 2013 (link); Gao et al., 2017 (link)). In brief, each sample, 20 mg of protein, was loaded on a 10% or 12% SDS-PAGE gel using a constant current and then transferred to PVDF membranes on a wet electrotransferring unit (Bio-Rad). Membranes were incubated with antibodies to IL-33 (1:200, R&D), ST2L (1:500, abcam), LC3B (1:3000, Abcam), Beclin-1 (1:1000, Bioworld), P62 (1:1000, Abcam), GRP78 (1:1000, Abcam), Cleaved-Caspase-3 (CC-3; 1:500, Bioworld), Bcl-2 (1:500, Abcam) and β-actin (1:10,000, Sigma). Then, the membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature. Immunoreactive proteins were detected with the ECL chemiluminescence system (Beyotime Institute of Biotechnology) and were obtained by Chemiluminescence Gel Imager. Films were automatically saved and densitometric analysis of the bands was performed with ImageJ.