Af 401 na

To analyse the expression of NLRP3 inflammasome, primary PMs were incubated for 3 h after seeding in the culture plate. Macrophages were assayed in opti‐MEM media (Gibco, 31985) in the presence or the absence of trehalose or saccharin 15 min prior to LPS stimulation (Invivogen, tlrl‐eklps; 10 ng/ml) for 3 h followed by adding ATP (Sigma–Aldrich, A6419; 5 mM) for 30 min to release IL‐1β. To confirm Atg7 protein deficiency in macrophage of Atg7^fl/fl or Atg7^fl/fl;Lyz2‐Cre mice, PMs were harvested, seeded, incubated for 3 h, and the adherent macrophages were collected for further analysis. Human T1R3 was analysed from cell lysates obtained from THP‐1 cell line or PBMCs, as described.⁸⁶ Proteins in the culture supernatant were concentrated by chloroform‐methanol precipitation, as described previously.⁹⁰ Primary antibodies used were goat anti‐mouse IL‐1β antibody (R&D Systems, AF‐401‐NA; 1:1000), mouse monoclonal anti‐NLRP3 antibody (Adipogen, AG‐20B‐0014; 1:1000), mouse monoclonal anti‐caspase‐1 antibody (AdipoGen, AG‐20B‐0042; 1:1000), rabbit anti‐Atg7 antibody (Cell Signaling, 2631; 1:1000), rabbit anti‐human T1R3 (abcam, ab229015; 1:1000), and mouse anti‐β‐Actin antibody (BD Biosciences, 612657; 1:5000).

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.