Ab 401 na

Sepsis was induced by CLP^{58 (link)} and compared to sham-operated controls. Briefly, mice were anesthetized with chloral hydrate (400 mg/kg body weight) and, through a midline celiotomy, the cecum was ligated distal to the ileocecal valve and punctured with an 18-gauge needle to allow feces to enter the peritoneal cavity. A control group was sham-operated. The mice were intravenously administered with either saline or other agents, including A438079 (80 mg/kg, P2RX₇ antagonist, Tocris Bioscience, Bristol, UK), anti-IL-1β mAb (100 μg/mouse, AB-401-NA, R&D Systems, Minneapolis, MN, USA) or anti-CXCL1 mAb (50 μg/mouse, AB-401-NA, R&D Systems), anti-CX3CL1 mAb (15 μg/mouse, AB-401-NA, R&D Systems), anti-CCL2/JE/MCP-1 mAb (10 mg/kg, AF-479-NA, R&D Systems) and anti-CXCL7/Thymus Chemokine-1 mAb (50 mg/kg, AF793, R&D Systems), at 1 h after CLP surgery. Normal Goat IgG (R&D Systems), Rat IgG2A isotype control (R&D Systems) were used as isotype control and administered using the same dosing schedule. For neutrophil depletion, mice were treated with intraperitoneal injection of 250 μg anti-granulocyte receptor-1 (Gr-1) mAb RB6-8C5 (BioXCell, West Lebanon, NH, USA) or an isotype control (BioXCell) 24 h prior to CLP surgery. Assessments were made by two independent observers who were blind to genotype and treatment status.

After stimulation, cells were washed once in PBS and lysed directly in SDS-PAGE gel loading buffer. Supernatants were diluted 1:1 in the same buffer. Samples were separated through 4–15% TGX precast gels (Bio-Rad) and transferred to nitrocellulose. The nitrocellulose blots were blocked with 5% nonfat dry milk and incubated overnight with antibodies against IL-1β (AB-401-NA; R&D Systems), caspase-1 (AG-20B-0042; Adipogen), Nlrp3 (AG-20B-0014; Adipogen), and Sting (50494; Cell Signaling Technology).