Asc d2w8u

Littermate WT and NLRP3^−/− mice were transcardially perfused with saline 24 hours after stroke. Brains were then removed and digested into a single-cell suspension and myelin removed (as described above), and cells were isolated by magnetic-activated cell sorting using CD11b+ magnetic beads (Miltenyi). Positive and negative CD11b+ cells were lysed in Tris-Triton buffer (150 mmol/L NaCl, 1% [vol/vol] Triton X-100, 50 mmol/L Tris, pH 7.5), supplemented with a protease inhibitor mixture. Cell lysates were separated by Tris-glycine SDS/PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes at 25 V using a semidry Trans-Blot Turbo system (Bio-Rad). Membranes were blocked in 2.5% (wt/vol) BSA in PBS, 1% (vol/vol) Tween 20 before being incubated with indicated primary antibodies at 4°C overnight. Membranes were then labeled with horseradish peroxidase–tagged secondary antibodies and visualized with Amersham ECL detection reagent (GE Healthcare). Western blot images were captured digitally using a G:Box Chemi XX6 (Syngene). Specific antibodies were used targeting mouse IL-1β (AF-401; R&D), ASC (D2W8U, Cell Signalling Technology), caspase-1 p10 (EPR16883; Abcam), and β-actin (Sigma).

A TUNEL assay kit (#11684817910) was purchased from Roche (Mannheim, Germany). Streptozotocin (STZ) was purchased from Sigma. An ROS assay kit (#S0033) was obtained from Beyotime (Shanghai, China). NLRP3 (ab214185), NeuN (ab104224) and IL-1β (ab9722) antibodies were purchased from Abcam (Cambridge, UK). ASC (D2W8U) and TXNIP (D5F3E) antibodies were obtained from Cell Signaling Technology (MA, USA). Caspase 1 (AF5418) and cleaved caspase-1 (AF4005) antibodies were purchased from Affinity.β-Actin and GAPDH antibodies were purchased from Bioss (Beijing, China). Genipin (Sigma-Aldrich, MO, USA) was diluted to appropriate concentrations in cell culture medium. ELISA kits were purchased from Abcam.

Antibodies recognizing the following proteins were used: AIM2 (63660; Cell Signaling), ASC (D2W8U; Cell Signaling), Caspase-1 (AG-20B-0042-C100; AdipoGen), GSDMD (ab209845; Abcam), HA (3F10; Roche), Flag (M2; Sigma-Aldrich), β-actin (AC-74; Sigma), GFP (clones 7.1 and 13.1, Roche). Antibody against MCMV IE1 (CROMA101) was from the Center of Proteomics, University of Rijeka, Rijeka, Croatia. Secondary antibodies coupled to horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch or DakoCytomation. HRP-coupled anti-Rabbit IgG heavy chain (ab99702) was purchased from Abcam. A secondary antibody coupled to AlexaFluor 488 was purchased from Invitrogen. The caspase-1 inhibitor VX-765 was from Invivogen. All antibodies and dilutions used in this study are listed in Supplementary Table 1.