P67phox

Heart samples were homogenated by lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, 1 µg/mL aprotinin leupeptin and pepstatin, 1 mM Phenylmethylsulfonyl fluoride (PMSF)] and then centrifuged at 12 000 rpm for 15 min at 4°C was collected, and the protein concentration was measured using the Bicinchoninic Acid (BCA) protein assay (Beyotime, China). Approximately 50 µg protein was loaded onto 10% or 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline and incubated with different primary antibodies at 4°C overnight. The following antibodies were used in this study: Bcl-2, Bax, p67^phox, P38 MAPK and phospho-P38 MAPK(Thr180/Tyr182), Jun NH2-terminal kinase (JNK) and phosphor-JNK (Thr183/Tyr185), ERK1/2MAPK and phosphor-ERK1/2 MAPK(Thr202/Tyr204) (Cell Signalling Technology, USA) used at a 1∶1,000 dilution and gp91^phox, p47^phox, p67^phox (Santa Cruz Biotechnology, CA) used at a 1∶200 dilution. The membrane was treated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. Blots were developed by ECL kit (Pierce Biosciences, USA). The intensity of protein band was semi-quantitative measured by image analysis software (GeneTools from SynGene).

Testicular tissue was homogenized in 1× radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling Technology, USA) and centrifuged at 12000 rpm for 15 min at 4°C. Protein concentrations in the supernatant were measured by a bicinchoninic (BCA) protein assay (Beyotime, China). An equal concentration of protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the following antibodies were used for western blotting: Bcl-2, Bax, p67^phox, p38 mitogen-activated protein kinase (MAPK), and phospho-p38 MAPK (Thr180/Tyr182); Jun NH2-terminal kinase (JNK) and phosphor-JNK (Thr183/Tyr185); extracellular signal-related kinase (Erk)1/2MAPK and phosphor-Erk1/2 MAPK (Thr202/Tyr204) (1:1000, Cell Signaling Technology, USA); and p47^phox and gp91^phox (1:200, Santa Cruz Biotechnology, USA). The membranes were then incubated with the appropriate secondary antibodies for 1 h at 37°C. Blots were visualized with enhanced chemiluminescence (ECL) kits (Pierce Biosciences, USA).

BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0.1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47^phox, P67^phox, gp91^phox, phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 4⁰ C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer’s protocol (eBioscience, CA).