Casp8

Cells were lysed using RIPA buffer supplemented with protease (ThermoFisher, 78,430) and phosphatase (ThermoFisher, 78,420) inhibitors, scraped, sonicated, and centrifuged (20,000 × g at 4 °C). Protein concentrations in the supernatant were determined using the Micro BCA Protein Assay kit (Thermo), and equal amounts of protein were resolved on pre-made Bis-Tris polyacrylamide gels (Life Technologies). Primary antibodies: pS345 CHEK1 (Cell signaling, #2348 L, 1:1000), pT68 CHEK2 (Cell signaling, #2197 S, 1:1000), pS139 H2A.X (Millipore, 05-636, 1:1000), clvd. Casp8 (Cell signaling, #8592, 1:1000), clvd. Casp9 (Cell signaling, #9502, 1:1000), clvd. Casp3 (Cell signaling, #9662, 1:1000), clvd. PARP (Cell signaling, #5625 P, 1:1000), and anti-actin (Cell Signaling Technology, 9470, 1:10,000). Primary antibodies were stored in 5% BSA (Sigma-Aldrich) and 0.1% NaN₃ in TBST solution. Anti-rabbit IgG HRP-linked (Cell Signaling Technology, 7074, 1:2500) and anti-mouse IgG HRP-linked (Cell Signaling Technology, 7076, 1:2500) were used as secondary antibodies. Chemiluminescent substrates (ThermoFisher Scientific, 34,077 and 34,095) and autoradiography film (Denville) were used for detection.

The brain tissues of mice were collected and lysed with RIPA solution. After electrophoresis, the samples were transferred to PDVF membrane, sealed with 5% skim milk for 1 h, and reacted with primary antibody (NLRP3, 1:1000, Casp8, 1:1000, Gsdmd, 1:1000, Trem2, 1:1000, Cleaved caspase-1, 1:1000, GAPDH, 1:1000, Cell Signaling Technology, USA) at 4 °C overnight. After cleaning, the samples were reacted with goat anti-mice IgG (1:3000; Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h.