Il 1β p17

Lung tissues were homogenized, separated on 12% SDS-PAGE gel, and then transferred onto a PVDF membrane. The membrane was blocked in 5% fat-free milk and probed with primary antibody against pro-IL-1β (Cell Signaling Technology, USA), IL-1β p17, NLRP3, caspase-1 p10 (Santa Cruz, USA) and IκB (Beyotime Biotechnology, China). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA; Cell Signaling Technology, USA) and enhanced chemiluminescence were applied to detect protein content. Images were collected using ChemiDoc XRS (Bio-Rad, USA). Bands were quantified using Image J.

Fetal bovine serum (FBS) was purchased from BioWhittaker Inc. (Walkersville, MO, USA). The following antibodies were purchased: NOX2, Rac1, and ANXA2 antibodies (BD Biosciences, Franklin Lakes, NJ, USA); NCF1 antibody (LifeSpan Biosciences, Seattle, WA, USA); p-JNK, JNK, PKCα^{Ser 657}, p-NF-κBp65, NF-κBp65, IL-1β p17, caspase-1 p10, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies (Jackson Immunoresearch, West Grove, PA, USA). 2′, 7′-dichlorofluorescein diacetate (CM-H₂DCFDA) was obtained from Invitrogen (Carlsbad, CA, USA). Methyl-β-cyclodextrin (MβCD), lipopolysaccharide (LPS), adenosine 5′-triphosphate disodium salt hydrate (ATP), 3-methyladenine, bafilomycin A1, N-acetyl-l-cysteine (NAC), rotenone, and 5-azacytidine were purchased from Sigma Chemical Company (St. Louis, MO, USA). The concentrations of all of pharmacological inhibitors listed did not show any significant cytotoxic effects by themselves as confirmed by FACS analysis in each experiment. All other reagents were of the highest purity commercially available and were used as received.