Biochemical Inhibition of NLRP3 Inflammasome

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)).