Sc 23460

Ipsilateral basal cortical samples facing blood clots were extracted. Western blotting was performed as described previously (25 (link)). Cortical samples were homogenized and centrifuged (1,000 × g, 10 min, 4°C). The supernatant was further centrifuged, and then, the protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein (50 μg) was suspended in loading buffer, denatured at 95°C for 5 min, and loaded on an SDS-PAGE gel. After being electrophoresed and transferred into polyvinylidene fluoride membranes, the membrane was blocked with nonfat dry milk buffer for 2 h and then incubated overnight at 4°C with the primary antibody for AIM2 (ab93015, 1:1,000, Abcam), pannexin-1 (ab124131, 1:1,000, Abcam), ASC (ab155970, 1:1,000, Abcam), caspase-1 (ab1872, 1:1,000, Abcam), P2X7R (1: 1,000, APR-004, Alomone, Jerusalem), IL-1β (SC-23460, 1:500; Santa Cruz), IL-18 (ab71495, 1:1,000; Abcam), and GAPDH (1:10,000, Fitzgerald, 10R-G109A). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein band densities were detected by x-ray film and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Ipsilateral basal cortical samples facing the blood clots were extracted. Western blot was performed as described previously^{37 (link)}. Briefly, cortical samples were homogenized, and centrifuged (1000 × g, 10 min, 4 °C). The supernatant was further centrifuged, and then, the protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein (50 μg) was suspended in loading buffer, then denatured at 95 °C for 5 min, and loaded on an SDS-PAGE gel. After electrophoresis and transfer onto polyvinylidene fluoride membranes, the membranes were blocked with non-fat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary antibodies for LC3 (2775, 1:1000, Cell Signaling), Atg5 (ab54033, 1:500, Abcam), Parkin (ab15954, 1:1000, Abcam), PINK1 (ab75487, 1:500, Abcam), NLRP3 (ab98151, 1:800, Abcam), ASC (ab155970, 1:1000, Abcam), caspase-1 (ab1872, 1:1000, Abcam), IL-1β (SC-23460, 1:500; Santa Cruz), IL-18 (ab71495, 1:1000; Abcam). The membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein band densities were detected by X-ray film and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The L4-L5 segments of the spinal cord were homogenized and subjected to SDS-PAGE as previously described48 (link). The membranes were incubated with the following primary antibodies at 4 °C overnight: goat anti-IL-1β (1:500; R&D), rabbit anti-caspase-1 (1:500; Abcam), rabbit anti-NALP1 (1:500; Abcam), rabbit anti-ASC (1:500; sc-22514-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mIL-1β (1:500; sc-23460; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-pSTAT3 (1:500; Cell Signaling Technology), rabbit anti-STAT3 (1:1000; 79D7; Cell Signaling Technology, USA), rabbit anti-pJAK2 (1:500; 3771; Cell Signaling Technology, USA), rabbit anti-JAK2 (1:1000; 3230; Cell Signaling Technology, USA), and mouse anti-β-actin (1:5000; 4967; Cell Signaling Technology, USA). The blots were then washed in TBST and incubated in the appropriate secondary antibody for 1 hour at room temperature. The secondary antibodies were donkey anti-goat lgG-HRP (1:10000; sc-2020; Santa Cruz Biotechnology, CA, USA), donkey anti-mouse lgG-HRP (1:5000; sc-2314; Santa Cruz Biotechnology, CA, USA), or HRP Affinipure Goat Anti-Rabbit lgG (H+L) (1:10000; E030120-01; ErthOx, CA, USA). The western blot images were captured on an ImageQuant LAS4000 mini image analyser (GE Healthcare, Buckinghamshire, UK), and the band levels were quantified using the Image J software, version 1.42q.