Hypersensitivity chemiluminescent substrate

We used lysis buffer to lyse the spinal cord tissue and primary cultured neurons. After protein quantification, electrophoresis, and Western transfer, we blocked the membranes with 5% bovine serum albumin followed by incubation with the appropriate primary antibodies as follows: NLRP3 (Abcam, 1:1,000), Caspase-1 (Abcam, 1:1,000), Caspase-1 (p20) (AdipoGen, 1:1,000), ASC (Abcam, 1:1,000), LC3 (CST, 1:2,000), and Beclin 1 (CST, 1:1,000), at 4°C overnight. Next, we incubated the membranes with secondary antibodies (Abgent, 1:30,000) followed by the hypersensitivity chemiluminescent substrate (Bio-Rad, United States). We obtained images using a MiVnt image analysis system (Bio-Rad, United States).

Spinal cord tissue and primary neurons were lysed by lysis buffer (Beyotime, Shanghai, China). After protein quantification, electrophoresis (12% SDS-PAGE gels), and Western transfer, the membranes were blocked with 5% BSA and incubated with the appropriate primary antibodies, such as HDAC1 (CST, 1:1,000), HDAC2 (Abcam, 1:800), HDAC3 (Abcam, 1:1,000), NLRP3 (Thermo Fisher Scientific, 1:1,000), caspase-1 (Proteintech, 1:800), and caspase-1 p20 (CST, 1:1,000) at 4°C overnight and then incubated with secondary antibodies (Abgent, 1: 20,000) followed by the hypersensitivity chemiluminescent substrate (Bio-Rad, Hercules, CA, United States), and protein bands were photographed by using a ChemiDoc™ MP imaging system (Bio-Rad).