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2 protocols using monosodium urate msu

1

Biochemical Inhibition of NLRP3 Inflammasome

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All chemicals and reagents were purchased from Sigma (St. Louis, MO, USA) and Invitrogen (CA, USA) unless specified otherwise. ATP, N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), U73122, BAPTA-AM, Glyburide, Bay 11-7082, and sp600125 were all from Sigma. Monosodium urate (MSU) was from Invitrogen. Gallein was from Tocris Bioscience (Bristol UK). U0126 was from Promega (Madison, WI, USA). SB203580 was from Cayman Chemical (Ann Arbor, MI, USA). z-YVAD-fmk (ALX-260-074) and Ac-YVAD-CHO (ALX-260-027) were from Enzo Life Sciences (Farmingdale, NY, USA). Muramyl dipeptide (MDP), Pam3CSK4, FLA-ST, and LPS-B5 Ultrapure were obtained from InvivoGen (San Diego, CA, USA). AZD9056 was from MedChemExpress (Princeton, NJ, USA). Monoclonal antibodies (mAbs) used for Western blotting included: anti-caspase-1 (D3U3E) and anti-human IL-1β (D7F10) were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-ASC (AL177) was from AdipoGen (San Diego, CA, USA); anti-NLRP3 (ALX-804-819) was from Enzo Life Sciences; Anti-β-actin mAb was purchased from BD Biosciences (San Jose, CA, USA). Anti-GAPDH mAb was from Proteintech (IL, USA). Abs used for cell stimulation (2A1 and mouse IgG1 control) and for the detection of signaling molecules have been described previously (24 (link)).
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2

Proteomic Analysis of NLRP3 Inflammasome

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Antibodies targeting the following proteins were used in this study: NLRP3 (sc-66846, rabbit polyclonal, Santa Cruz Biotechnology, USA; ab4207, goat polyclonal, Abcam, USA), TRX (ab26320, rabbit polyclonal/ab16965, mouse monoclonal, Abcam, USA), TXNIP (sc-166234, mouse monoclonal, Santa Cruz Biotechnology, USA; ab215366, rabbit polyclonal, Abcam, USA), COX IV (ab14744, mouse monoclonal, Abcam, USA), Fibronectin (FN) (ab45688, rabbit monoclonal, Abcam, USA), Caspase-1 (ab1872, rabbit polyclonal, Abcam, USA), IL-1β (ab9722, rabbit polyclonal, Abcam, USA), IL-18 (#10663-1-AP, rabbit polyclonal, Proteintech, USA), Collagen I (M38, mouse monoclonal, DSHB Biosciences, USA; ab23446, mouse monoclonal, Abcam, USA), and Collagen IV (ab6586, rabbit polyclonal, Abcam, USA). MitoQ was obtained from FOCUS Biomolecules, and monosodium urate (MSU), TXNIP siRNA, TRX siRNA, DHE, MitoTracker Red, MitoSOX Red and DCFDA were purchased from Invitrogen.
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