Nb120 10454

BMDMs were lysed in RIPA buffer and sample loading buffer containing SDS and 100 mM DTT. Proteins were separated on 10–12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes, membranes were blocked in 5% skim milk and incubated with primary antibodies against caspase-1 (Adipogen, AG-20B-0042), caspase-11 (NB120-10454, Novus), anti-β-actin (#8457; Cell Signaling Technology) followed by secondary anti-rabbit or anti-mouse HRP antibodies (Jackson ImmunoResearch Laboratories).

zVAD, HG-9-91-01, and YKL were purchased from MedchemExpress. Necrostatin-1 was from Calbiochem. GSK’872 and SM-164 were from Selleck. Protein A/G Magnetic Beads were purchased from Pierce. Lipofectamine 2000 was from Life Technologies. LPS and poly I:C were purchased from InvivoGen. Mouse and human recombinant TNF were purchased from Novoprotein. Propidium iodide (PI) and cycloheximide (CHX) was from Sigma. Human recombinant RIPK1 and RIPK3 were from AtaGenix. Elisa kits for TNF and IL-1β were purchased from R&D. ADP-Glo kinase assay kit was from Promega. Immunoblotting was performed with following antibody: anti-RIPK1 (610459, BD Biosciences); anti-hRIPK1 S166 (44590, Cell Signaling); anti-mRIPK1 S166 (31122, Cell Signaling); anti-hRIPK3 (13526, Cell Signaling); anti-mRIPK3 (2283, ProSci); anti-hRIPK3 S227 (91702, Cell Signaling); anti-mRIPK3 T231/S232 (91702, Cell Signaling); anti-hMLKL (ab184718, Abcam); anti-mMLKL (AP14272b, ABGENT); anti-hMLKL S358 (ab187091, Abcam); anti-mMLKL S345 (ab196436, Abcam); anti-GAPDH (sc-32233, Santa Cruz); anti-cleaved Caspase-8 Asp387 (8592, Cell Signaling); anti-cleaved Caspase-3 (9661, Cell Signaling); anti-GSDME (ab215191, Abcam), anti-GSDMD (ab209845, Abcam), anti-Caspase-1 (AG-20B-0042-C100, AdipoGen), anti-Caspase-11 (NB120-10454, Novus).

BMECs were cultured briefly on cover glasses (pre-treated with poly-L-lysine, 0.1 mg/mL, Sigma-Aldrich) and incubated with histone (50–200 μg/mL) for 16 h. The cells were fixed with 4% (w/v) paraformaldehyde for 30 min and washed three times. After that, the samples treated with antigen retrieval, non-specifically blocked with 3% H₂O₂ in methanol for 40 min, 5% goat serum/PBS blocked for 45 min and followed by incubation with antibody to caspase 1 (2.5 μg/mL, AB1872, Abcam), caspase 3 (2.5 μg/mL, AB4051, Abcam), caspase 11 (2.5 μg/mL, NB120-10454, Novus), and NLRP3 (0.5 μg/mL, Boster, China) overnight at 4°C and incubated with the secondary antibody (Maxim, KIT9710, China) at room temperature for 30 min. Finally, the samples were visualized with DAB (Maxim, DAB-0031, China) and counterstained with hematoxylin.