Caspase 1 p20

Antibodies against NLRP3 (Cell Signaling) or caspase-1 p20 (AdipoGen) were incubated with protein G Sepharose (GE Healthcare) overnight at 4°C. After the redundant antibodies were washed out, the whole cell proteins (400 μg) or cell culture supernatants (1 mL) were incubated with the complex overnight at 4°C. The resultant immune complexes binding to protein G Sepharose were washed thoroughly five times with ice-cold Dulbecco's phosphate-buffered saline supplemented with 0.1% Nonidet P-40. The immunoprecipitated proteins were eluted and denatured by boiling them at 95°C for 5 min in the sample buffer and separated as described above; subsequently, the amounts of intracellular NLRP3 or secreted caspase-1 p20 were analyzed by western blotting.

Lung tissues were homogenized in RIPA in the presence of a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and Phosphatase Inhibitor Cocktail (Roche). The protein concentrations were determined by a BCA kit (Pierce, Rockford, IL, USA). Equal concentrations of protein (25 μg) were separated by SDS-PAGE on 12% acrylamide gels. The primary antibodies used were as follow: NLRP3 (Adipogen, San Diego, CA, USA), caspase 1 (p20) (Adipogen), IL-1β, pro-IL-1β (R&D Systems, Minneapolis, MN, USA), TLR2 (Millipore, Billerica, MA, USA), AANAT (Sigma), ASMT (Abcam, Boston, MA, USA) and GAPDH (KANGCHEN Biotech, Shanghai, China) for 1 h at 37°C, followed by at 4°C overnight. Blots were washed thrice with TBST and probed with appropriate secondary antibodies for 1 h at room temperature. After washing three times, the immune-reaction was analyzed using the ECL detection system (Pierce). The band density was determined by ImageJ software (NIH, Bethesda, MD, USA).

The A. sobria–infected PMφs pellets (MOI=1, 10, 100) were lysed in RIPA buffer containing 1 mM phenylmethylsulfonylfluoride (PMSF, Solarbio, Beijing). The lysed PMφ pellets and A. sobria–infected PMφ supernatants were concentrated using methanol and chloroform as previously described (Wang et al., 2018a (link)) and quantified using BCA method with BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein extraction (30 μg) were separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane (Millipore, USA). The protein-contained membranes were blocked in 5% non-fat milk; incubated at 4°C overnight with primary antibodies, including IL-1β (1:2,000, R&D, USA), caspase-1 (p20) (1:1,000, Adipogen, Switzerland), NLRP3 (1:1,000, Adipogen, Switzerland), ASC (1:500, Wanleibio, Shenyang), and β-actin (1:5000, Proteintech, Wuhan); incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary detection antibodies, including rabbit anti-goat IgG, goat anti-mouse IgG (H+L), and goat anti-rabbit IgG (1:2,000, Proteintech, Wuhan); and visualized with Immobilon Western Chemiluminescent HRP substrate (Millipore, USA) on a ChemiScope Western Blot Imaging System (Clinx, Shanghai).

Proteins from lung tissues were extracted and analyzed by Western blotting. Total proteins were extracted with cell lysis buffer (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Proteins were separated by electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Germany). Membranes were then blocked with 5% bovine serum albumin and incubated with primary antibodies against NLRP3 (AdipoGen, USA, clone: Cryo-2, dilution: 1:1000), Caspase-1 p20 (AdipoGen, USA. clone: Casper-1, Dilution: 1:1000), pCaMK4 (dilution 1:1000) at 4°C overnight. GAPDH (Cell Signaling Technology, USA, clone: 14C10, dilution 1:2000) was used as internal control. Membranes were washed and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (both from Cell Signaling Technology, USA, dilution: 1:2000). Signals were detected with enhanced chemiluminescence analysis kit (Cell Signaling Technology, USA).

Proteins were extracted from lung tissues and BALF AMs using a RIPA reagent containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Equal amounts of proteins were separated on 12% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were incubated with 5% non-fat milk for 1 h and then with primary antibodies overnight at 4 °C. The following primary antibodies were used: Irg1 and COX IV were obtained from Abcam (Boston, MA, USA), NLRP3 and Caspase 1(p20) were from Adipogen (San Diego, CA, USA), IL-1β (R&D Systems, Minneapolis, MN, USA), Nitrotyrosine, SOD2, OPA1, Mfn2, DRP1, Fis1, IL-4, IL-5 and IL-13 were all from Santa Cruz (CA, USA) and GAPDH (KANGCHEN Biotech, Shanghai, China). For the quantitative analysis, the intensity of the protein bands was determined by using Image J 1.38x software (NIH, Bethesda, MD, USA).

BMDMs infected with bacteria were dissolved by lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 mM Na₃VO₄, 0.1 mM NaF, 1 mM PMSF, 5 mg/mL Aprotinin, 5 mg/mL Leupeptin, and 5 mg/mL Pepstatin A. Lysate and supernatant was harvested and used for immunoblotting analysis. The lysate (30 mg) were subjected to 12% sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 5% milk, the membrane was blotted with antibodies against PARP (Cell Signalling), pMLKL (Abcam), RIPK3 (Abgent), Caspase-1 p20 (Adipogen), Cleaved caspase-3 (Cell Signalling), AIM2 (ebioscience), and β-Tubulin (Sungene Biotech, Tianjin, China).