Cleaved il 1β asp117

LPS and nigericin were purchased from Sigma-Aldrich (St. Louis, MO). NF-kB inhibitor, QNZ (EVP4593), was supplied by MedChemExpress (New Jersey, USA). Antibodies against FTO, NLRP3, FoxO1, P65, p-P65, IL-1β and Cleaved-IL-1β (Asp117) were obtained from Cell Signaling Technologies (Beverly, MA). ELISA kits of IL-1β, interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin-12(p70) (IL-12(p70)) were purchased from eBioscience (San Diego, CA). Brilliant Violet 421™ anti-mouse F4/80 antibody, PE anti-mouse/human CD11b antibody, FITC anti-mouse I-A/I-E antibody, APC anti-mouse CD80 antibody, PE/Cy7 anti-mouse CD86 antibody, FITC anti-mouse Ly-6G antibody and APC anti-mouse CD40 antibody were obtained from BioLegend (San Diego, CA, USA). Lipidoid (C12-200) was supplied by Xinjiahecheng Medical Chemistry Corporation (Wuhan, Hubei, China). mPEG2000-DEG was purchased from NOF Corporation (Tokyo, Japan).

Cells were lysed in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology) supplemented with Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Life Technologies). Cell lysates prepared for SDS-PAGE using NuPAGE™ LDS sample buffer and NuPAGE™ sample reducing agent (Invitrogen). Supernatant samples from cell-free supernatants were prepared using sample buffer containing SDS and Dithiothreitol (DTT). The primary antibodies were used against caspase-1 (p20) (mouse; Adipogen #AG-20B-0042-C100), caspase-1 (p20) (human; Adipogen #AG-20B-0048-C100), cleaved-IL-1β (Asp117) (Cell Signaling Technology #52718), IL-1β (R&D Systems #AF-401), Ras (Abcam #52939), active Rac1 (NewEast Biosciences #26903). β-actin (Cell Signaling Technology #4970, or Santa Cruz Biotechnology #sc-47778) or Vinculin (Cell Signaling Technology #13901) were used as loading controls. As a secondary antibody, horseradish peroxidase (HRP)-linked anti-rabbit IgG or anti-mouse IgG were used (#7074 and #7076S Cell Signaling Technology). The blot signals were detected using WesternBright Quantum HRP substrate (Advansta), imaged using ChemoCam Imager 3.2.0 (Intas Science Imaging Instruments GmbH) and quantified using ImageJ (NIH) software.