Wl01980

The protein extracts of brain tissue were prepared as described previously. To determine NLRP3, ASC, and cleaved caspase-1 protein expressions in the ischemic core of the brain (Fig. 2A), the protein concentrations were measured using BCA assay. Protein samples were separated by SDS-PAGE and were transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% nonfat milk in TBST (0.1% Tween 20 in TBS) for 2 hours at room temperature and incubated with primary antibodies of anti-NLRP3 (1:1000, A12694; ABclonal Technology, Wuhan, China), anti-ASC (1:1000, A16672; ABclonal Technology, Wuhan, China), anti-cleaved caspase-1 (1:1000, WL03325; Wanleibio, Shenyang, China), and anti-NF-κB (1:500, WL01980; Wanleibio, Shenyang, China) at 4°C overnight. After being washed 3 times, the membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) secondary antibodies (1:5000, AB0101; Abways Technology, Shanghai, China) for 2 hours. Then, the blots were visualized by enhanced chemiluminescence detection system (Chemidoc XRS+, Bio-Rad; Berkeley, CA). The results were normalized to anti-β-actin (1:1000, AY0573; Abways Technology, Shanghai, China) or anti-H3 (1:500, WL09804a; Wanleibio, Shenyang, China) and the intensity of bands was quantified by ImageJ.

Firstly, BCA assay kit (WLA004, Wanleibio, China) and different concentrations of SDS-PAGE gels (WLA013, Wanleibio, China) were used to quantify the proteins in the brain homogenates or BV-2 cell lysates, which were then transferred to PVDF membranes (IPVH00010, Millipore, USA). Secondly, membranes were blocked with 5% non-fat dry milk (Q/NYLB 0039S, Yili, China) in TBST (T1081, Solarbio, China) and incubated with primary antibodies against galectin-3 (1:500, 60207-1-Ig, Proteintech, China), toll-like receptor 4 (TLR4, 1:400, WL00196, Wanleibio, China), nuclear factor kappa-B (NF-ĸBp65, 1:500, WL01980, Wanleibio, China), caspase-1 (1:500, WL03450, Wanleibio, China), interleukin-1β (IL-1β, 1:1000, WL00891, Wanleibio, China), NLRP3 (1:1000, MA5-23919, Thermo Fisher, USA), and β-actin (1:400, WL01372, Wanleibio, China), and then washed with TBST and incubated with HRP-IgG (1:5000, goat anti-rabbit, WLA023, Wanleibio, China). Lastly, ECL kit (WLA003, Wanleibio, China) was used to detect immunoreactive bands, and Gel-Pro-Analyzer software (WD-9413B, Beijing Liuyi, China) was used to scan and analyze bands. β-actin was set as internal standard to normalize film signals, and signals of sample in Control group were normalized to 1.0.