Nlrp3

Peritoneal macrophages which were treated with stimulation were lysed in immunoprecipitation (IP) buffer, namely, a protease inhibitor cocktail, and then the cell lysates were incubated overnight at 4°C with the specific antibodies. The next day, immunoprecipitates were washed by IP buffer 4 times and incubated with Protein A/G plus-agarose(Santa Cruz sc-2003) at 4°C for 4 h. At last, immunoprecipitates were boiled with 1% (w/v) SDS sample buffer. The proteins bound by antibody were precipitated by protein A/G beads and subjected to immunoblotting analysis. The proteins were separated by SDS-PAGE, and then we have translated the proteins onto PVDF membranes for immunoblot analysis. After that, the membranes were blocked with 5% dried milk in TBS-T (50 mM Tris/HCL, 150 mM NaCl, pH 7.6 and 0.1% Tween-20) for 1 h at room temperature. After blocking, PVDF membranes were incubated with various primary antibodies such as human IL-1β (Abcam ab9722), human GSDMD (Abcam ab210070), Caspase-1 (Abcam ab179515), IL-1β (RD systems AF-401-NA), NLRP3 (Adipogen), ASC (Adipogen AL177), GSDMD (Abcam ab133514) and NEK7 (Abcam ab133514) at 4°C overnight. The next day, membranes were washed with TBS-T and incubated in corresponding horseradish peroxidase-conjugated secondary antibodies (1:5,000, KPL) for 1 h at room temperature.

Kidneys were washed with PBS and lysed in an ice-cold lysis buffer containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Proteins were separated with 10% PAGE and electroblotted onto a PVDF membrane (Millipore, Madrid, Spain). The membrane was incubated with primary antibody raised against NLRP3 (1:1000, AdipoGen, San Diego, USA), ASC, caspase-1, IL-1β, MAVS, LC3, parkin, PINK1, HIF-1α, AIF, cytochrome c, Bax, prohibitin, α-SMA (1:1000, Abcam, Cambridge, UK), GAPDH (1:1000, cell signaling MA, USA), β-actin (1:1000, Santa Cruz, CA, USA) and, subsequently, with horseradish peroxidase-conjugated goat anti-rabbit or mouse immunoglobulin G (1:10000, Santa Cruz, CA, USA). The immunoreactive bands were detected by chemiluminescence (ECL, BioFX Laboratories, Inc. MD, USA). β-actin and GAPDH were used as internal controls of tubular cells and renal tissues. Prohibitin and GAPDH was used for the internal control of mitochondria and cysotol, respectively.

Protein extraction from each sample was performed using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The protein concentration was determined by a BCA kit (Beyotime,Biotech, Shanghai, China). Equivalent amounts of total protein were separated on an SDS−PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% nonfat milk at RT for 1h. Then, the PVDF membranes were incubated with NLRP3 (Adipogen, Inc), Caspase 3 (Abcam, Inc.), GPX4 (Abcam, Inc), PFKFB3 (Abcam, Inc.), HK2 (Abcam, Inc), GLUT1 (Huabio,Inc) and β-actin (Cell Signaling Technology, Inc) overnight at 4°C. The blots were washed and incubated with an HRP-conjugated secondary antibody at 37°C for 1 hour. Finally, the membranes were washed twice and detected by a chemiluminescence kit. Protein quantification was carried out using ImageJ software (NIH) and relative to β-actin expression.

Cells and culture supernatants, and tissue homogenates were incubated with cell lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40) and denatured in Laemlli buffer by boiling for 10 min. Proteins were separated by SDS-PAGE electrophoresis (Thermo Scientific) after which proteins were transferred to nitrocellulose membranes (Thermo Scientific) using semi-dry (20 min) or turbo (7 min) blotting. Blocking and antibody incubation were performed in PBS or TBS supplemented with 0.05% Tween20 (vol/vol) and 3% or 5% (wt/vol) non-fat dry milk. The membranes were incubated overnight at 4°C with primary antibodies against Caspase-1 (1:1000; Adipogen), IL-1β (1:2000; GeneTex), Nlrp3 (1:1000; Adipogen), Caspase-11 (1:1000; Novus biologicals), GSDMD (1:1000)[29 (link)], ASC (1:1000; ECM Biosciences), MLKL (1:1000; Millipore), or Caspase-3 (1:1000; Cell signaling). After washing, membranes were incubated with HRP-conjugated anti-mouse, anti-rabbit or anti-rat secondary antibodies (1:5000; Jackson ImmunoResearch Laboratories, 111-035-144, 112-035-143, 112-035-143) or were incubated with the directly labeled primary antibody β-actin-HRP (1:10000; Santa Cruz) for up to 3 h. Proteins of interest were detected by the enhanced SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

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 cell-free supernatants were extracted by methanol/chloroform precipitation as described previously42 (link). Cell extracts were also prepared. Samples were separated on SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin for 2 h and then incubated overnight at 4 °C with antibodies specific for phosphor-Thr466 on PKR (Abcam), total PKR (Abcam), caspase-3 (Cell Signaling), BAX (Abcam), IL-1β (Santa Cruz), caspase-1 (Santa Cruz), NLRP3 (Adipogen), STAT-2 (Santa Cruz), ADAR1 (Santa Cruz) and GAPDH (Sigma) followed by incubation with HRP-conjugated secondary antibody. The quantitative relative expression of proteins were calculated by normalizing the densitometric analysis to GAPDH.