Sds page gel

The hippocampuses was promptly dissected out on ice and homogenized in ice-cold RIPA buffer containing a protease inhibitor cocktail for 30 minutes. After centrifugation for 10 min at 4°C and 12,000 rpm, supernatants were divided into eppendorf tubes and stored at-20°C until required for protein assay, which was performed using Pierce BCA protein assay kits (Thermo scientific, USA). For western blot analysis, mixed one part of sample loading buffer (Applygen Tech Inc., China) with 4 parts of tissue protein and then boiled the mixture at 100°C water bath for 10 minutes. Denatured proteins were separated by 10% SDS-PAGE gel (CWBIO, China) electrophoresis for 1 h at 80 V then shifted to 100 V for another 2 h. Thereafter transferred to a 0.45 μmpolyvinylidene-difluoride (PVDF) membranes for 50 minutes at 100 V. Membranes were then washed for 3 × 10 min in 0.1% Tween-20 PBS (TBST) between each of following steps: 1 h block in 5% non-fat milk, over-night incubation at 4°C with primary antibodies. The immunoreactive bands were visualized by using secondary antibodies and ECL chemiluminescence detection kit (CWBIO, China).

Culture supernatant was harvested to collect dead/floating cells by centrifugation at 12,000 rpm for 10 minutes at 4°C and pooled with adherent cells washing with PBS buffer three times. All cells were lysed on ice to extract protein. Equal amounts of protein lysates (40ug) were separated on SDS-PAGE gels (CWBIO, China) under reducing conditions and subsequently transferred onto nitrocellulose membranes. The caspase-1 antibody (1:1000 dilution), caspase-11 antibody (1:1000 dilution), GSDMD antibody (1:1000 dilution), GSDME antibody (1:1000 dilution), cHSP60 antibody (1:200 dilution), caspase-8 antibody (1:1000 dilution), caspase-8 p18 antibody (1:1000 dilution), β-Tubulin antibody (1:2000 dilution) and GAPDH antibody (1:2000 dilution) were incubated with polyvinylidene difluoride membranes at 4°C overnight. Then we washed the blots with PBS and incubated them with the previous described secondary antibodies. Protein bands were visualized using ECL detection regents (GE Healthcare, USA). Western blot quantification was performed by measuring band intensity with ImageJ freeware (National Institutes of Health, Bethesda, MD, USA). The quantification of cleaved caspases band was normalized first to GAPDH then to the pro-caspase band intensity. The quantification of GSDMD-NT band was normalized first to GAPDH and then to the full-length GSDMD intensity.

After removing the supernatant, the cells were washed twice with PBS at 4°C. RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor (Sigma–Aldrich, USA) and 1% serine protease inhibitor (Sigma–Aldrich, USA) was added for 30 min to lyse cells and extract proteins from each sample. The concentrations of total proteins were detected by a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (ThermoFisher, USA) at a ratio of 4:1 and boiled at 99°C for 10 min. Samples were loaded and run on SDS–PAGE gels (CWBIO, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 2% skim milk (BD, USA) for 1 h at room temperature and then incubated with anti-IKKα, anti-IKKβ, anti-pIKKα/β, anti-p65, anti-pp65, anti-p38, anti-pp38, anti-SAPK/JNK, anti-pSAPK/JNK, and anti-GAPDH primary antibodies (Abcam, UK) overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit or goat anti-mouse HRP (Abcam, UK) for 1 hour. Membranes were washed and exposed to chemiluminescent HRP substrate (Millipore, USA). Images were obtained using the GeneGnome XRQ system (Syngene, USA) and analyzed using ImageJ software.