Total p 65

Protein samples were harvested from penumbra. Tissues were ground separately in RIPA buffer comprising protease and phosphatase inhibitors (cocktails and PMSF from Aspen) for 30 min at 4°C. A BCA kit (Aspen) was used to detect the total protein concentration of each sample. Proteins were processed by SDS-PAGE (10–12.5%) and electro-blotted onto a PVDF membrane. And the membrane was then incubated in blocking buffer (5% skim milk) for 1 h at room temperature and incubated with primary antibodies including GSDMD (Abclonal), NLRP1, NLRP3, Caspase-1 (Novus), IL-1β,IL-18 (R&D), total p-65 (Proteintech),phosphorylated p-65 (Abclonal), GAPDH (Proteintech) overnight at 4°C. After washing three times, the membrane was incubated in secondary antibody for 1 h at 24°C. The proteins were scanned with a Bio-Rad system. ImageJ software was used to quantify protein levels which were normalized to GAPDH (n = 6/group).

Primary antibodies against phospho-AKT (S473), pan-AKT, phospho-FoxO3a (S253), totalFoxO3a, phospho-p65 (S276), total-p65, cleaved-caspase3, cleaved-caspase1, cleaved-PARP1, LC3 and p62 were purchased from Cell Signaling Technology (Boston, Massachusetts, USA), total-caspase3, PAR and p-MLKL(S358) was purchased from abcam (Cambridge, UK), p53, PARP1, AIF, pan-AKT, totalFoxO3a, total-p65, GPX4 and β-actin were purchased from proteintech (Wuhan, Hubei, China), Cox IV, H3 and HRP conjugated anti-rabbit/mouse secondary antibodies were purchased from Abbkine (Wuhan, Hubei, China). Lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). AKT inhibitor SC66, TIC10, autophagy inhibitor CQ, ULK101, pan-caspase inhibitor Z-VAD-FMK and PARP1 inhibitor 3AB were purchased from Selleck (Houston, Texas, USA). CCK-8 kit was from 7 Sea Biotech (Shanghai, China). ROS inhibitor NAC was purchased from Topscience (Shanghai, China).