Western Blotting

Protein extracts were prepared from longitudinally halved mouse brain (~200mg) in 500μL homogenization buffer consisting of 10 mM Tris, pH 7.4, 100mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, ​0.5% deoxycholate, and 1× complete protease inhibitor cocktail (Roche, 11836153001). Debris were removed by centrifugation at 18,000 × g for 5 min at 4°C. The supernatant fraction was quantified through use of the Bradford assay (Bio-Rad, 5000006). For each sample, 50 μg of total protein extracts was resolved by 12% SDS-PAGE and transferred onto a PVDF membrane for immunodetection. The membrane was incubated with antibodies specific to CtBP1 (BD Biosciences, 612042, 1:1,000), CtBP2 (BD Biosciences, 612044, 1:1,000), S100A9 (Santa Cruz Biotechnology, sc-58706, 1:1,000), NLRP3 (Santa Cruz Biotechnology, 134306, 1:1,000) and GAPDH (Sigma, G8795, 1:5,000) at 4°C overnight, followed by peroxidase-labeled appropriate secondary antibodies at room temperature for 1h. The membrane was developed using an enhanced chemiluminescence substrate (Millipore Corporation, WBKLS0500) and scanned with a ChemiDoc MP imager (Bio-Rad). Raw signal intensity for each band was measured using Image J software (4.0.1 version).