Western Blot Analysis of Aeromonas sobria-Infected Macrophages

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).