Ab207323

Brain or spleen tissue were washed with PBS and lysed in 1× RIPA buffer with protease and phosphatase inhibitors. Protein concentrations were determined by the Bradford assay, and equal amounts of protein were subjected to SDS/PAGE. Then, the samples were transferred onto nitrocellulose membranes. The blots were incubated overnight at 4 °C with the following antibodies: GSDMD (1:1000, ab209845, Abcam, Cambridge, England), Caspase-1 (1:1000, NBP1-45433, NOVUS, Colorado, USA), Caspase-11 (1:1000, ab180673, Abcam, Cambridge, England), IL-1β (1:1000, #31202, CST, Southcarolina, USA), IL-18 (1:1000, ab207323, Abcam, Cambridge, England) and α-tubulin (1:1000, #9099, CST, Southcarolina, USA). The membranes were incubated with appropriate secondary antibodies for 2 h at room temperature. The blots were visualized with SuperSignal West Femto substrate (Pierce) on a ChemiDoc Imaging System (Bio-Rad) and analyzed by ImageJ.

The cell lines used were as follows: HAE (CP-H209, Procell, Wuhan, China), A549 (CL-0016, Procell), HCC827 (CL-0094, Procell), NCL-H1299 (CL-0165, Procell), and NCL-H524 (CL-0403, Procell).
The following antibodies were used: anti-TMED2 (ab251705, Abcam, Cambridge, USA), anti-Ki-67 (ab15580, Abcam), anti-CEA (ab207718, Abcam), anti-NSE (ab180943, Abcam), anti-EGFR (ab200828, Abcam), anti-TLR4 (ab13556, Abcam), anti-NF-κB-p-p65 (BM3940, Boster, Wuhan, China), anti-IL-1β (ab2105, Abcam); anti-IL-18 (ab207323, Abcam); and anti-β-actin (M01263-2, Boster). The secondary antibodies used were anti-rabbit IgG (AS014, ABclonal, Wuhan, China) and anti-mouse IgG (H+L) (AS003, ABclonal).

The total proteins were extracted from the hippocampal region of mice, and the protein concentration was determined by BCA assay. Equivalent amounts of protein samples were sampled on a 10% SDS-PAGE gel for electrophoretic separation of the proteins, and then, the PVDF membranes were electrotransformed by constant current at 280 mA for 90 min. Subsequently, the membranes were soaked with 5% skim milk powder in TBST (Tris-buffered saline containing 1% Tween-20) for 2 h. Then, the corresponding primary antibody was added overnight at 4°C. The primary antibodies were NLRP3 (1: 1000, ab263899), Caspase-1(1: 1000, ab179515), IL-1β (1: 1000, ab234437), IL-18 (1: 1000, ab207323), and β-actin (1: 2000, BL005B) and were purchased from Abcam (Cambridge, UK) except β-actin which was purchased from Biosharp (Anhui, China). Then, membranes were removed, rinsed 3x for 10 minutes with TBST, and incubated for 2 h at 37°C with the goat anti-rabbit IgG (1 : 10000, BL003A, from Biosharp, Anhui, China), respectively. TBST was used to wash the membranes three times before they were subjected to Bio-Rad electrophoresis (Bio-Rad Laboratories, Hercules, CA, United States). Analyzing all band intensities was done using ImageJ software.

Chrysomycin A was provided by Prof. Hua-Wei Zhang (Zhejiang University of Technology). Dulbecco’s modified Eagle’s medium (DMED) and fetal bovine serum (FBS) for cell culture were purchased from Gibco BRL (Grand Island, NY, USA). LPS (Escherichia coli 0127: B8) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits IL-1β (EM001), IL-6 (EM004), TNF-α (EM008), MCP-1 (EM018) and GM-CSF (EM020) were purchased from ExCell Biology (Shanghai, China). IL-17 (PI545), and CXCL12 (PC201) were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against COX2 (ab15191) and IL-18 (ab207323) were purchased from Abcam (Cambridge, UK). Antibodies for β-actin (3700), NLRP3 (15101), cleaved caspase-1 (89332) and IL-1β (12242) were purchased from Cell Signaling Technology (Beverley, CA, USA).