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3 protocols using anti human cleaved il 1β

1

Inflammasome Activation Reagents Protocol

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Tanshinone I (Tan I, HY-N0134), MCC950 (HY-12815 A), and nigericin (HY-127019) were from MedChemExpress. ATP, SiO2, Pam3CSK4, poly (I:C), poly (dA:dT), and ultrapure lipopolysaccharide (LPS) were from Invivogen. Phorbol 12-myristate 13-acetate (PMA, P8139) and DMSO (D2650) were from Sigma Aldrich. The Starfect high-efficiency transfection reagent (C101-10) was from GenStar. Antibodies were used as follows: anti-mouse IL-1β (1:1000, AF-401-NA, R&D), anti-mouse caspase-1 (1:1000, AG-20B-0042, Adipogen), anti-human cleaved IL-1β (1:2000, 12242, Cell Signaling Technology), anti-human caspase-1 (1:2000, 4199 S, Cell Signaling Technology), anti-NLRP3 (1:2000, AG-20B-0014, Adipogen), anti-ASC (1:1000, sc-22514-R, Santa Cruz), anti-Flag (1:2000, 20543-1-AP, Proteintech), and anti-GAPDH (1:5000, 60004-1-Ig, Proteintech).
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2

NLRP3 Inflammasome Activation Assay

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MSU, nigericin, ATP, poly(deoxyadenylic-thymidylic) acid sodium salt (poly (dA:dT)), polyinosinic:polycytidylic acid (poly (I:C)), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO), and ultrapure LPS were purchased from Sigma–Aldrich (Munich, Germany). Pam3CSK4 was obtained from InvivoGen (Toulouse, France). MCC950 and CDN were from TargetMol (Boston, MA, USA). Lfn-Flic was kindly provided by Dr. Tao Li from National Center of Biomedical Analysis. Anti-mouse CASPASE-1 (1:1000, AG-20B-0042) was bought from Adipogen (San Diego, CA, USA). Anti-human cleaved IL-1β (1:2000, 12242), anti-human CASPASE-1 (1:2000, 4199 S), anti-mouse IL-1β (1:1000, 12507), anti-NLRP3 (1:2000, 15101S) were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (1:2000, 60004-1-1g) was purchased from Proteintech (Chicago, IL, USA).
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3

Inflammasome Activation Assay

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Nigericin, ATP, poly(dA:dT), poly(I:C), phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO) and ultrapure LPS were obtained from Sigma-Aldrich (Munich, Germany). Silicon dioxide (SiO2) and Pam3CSK4 were obtained from InvivoGen (Toulouse, France). MCC950, carnosol, and geldanamycin (GA) were obtained from TargetMol (Boston, MA, USA). MitoTracker and MitoSOX were manufactured by Invitrogen (Carlsbad, CA, USA). Salmonella was a gift from Dr. Tao Li of the National Center of Biomedical Analysis. Anti-mouse Caspase-1 (1:1000, AG-20B-0042) was from Adipogen (San Diego, USA). Anti-human cleaved IL-1β (1:2000, 12242), Anti-mouse α-SMA (1:1000, 19245s), anti-human Caspase-1(1:2000, 4199S), anti-mouse IL-1β (1:1000, 12507) and anti-NLRP3 (1:2000, 15101S) were from Cell Signaling Technology (Boston, USA). Anti-ASC (1:1000, sc-22514-R) was from Santa Cruz Biotechnology (Dallas, USA). Anti-DDDK tag (1:3000, 20543–1-AP), Anti-HSP90 (1:3000, 13171-1-AP) and Anti-GAPDH (1:2000, 60004-1-1G) were from Proteintech Group (Chicago, USA).
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