Deae 52

AAP was extracted as described by Zeng et al. (21 (link)). with slight modifications. Briefly, A. auricular powder (20 g) was extracted with 1000 mL deionized water for 2 h at 90°C. The total extraction solution was concentrated under reduced pressure to 1/3 of the original volume and then precipitated with three volumes of chilled 95% ethanol at 4°C overnight. Protein content was removed by applying the Sevag reagent (chloroform : n-butyl alcohol = 4:1, v/v) to the precipitate solution. The aqueous solution was dialyzed against deionized water for 3 days (7 kD), and the water was changed three times a day. Crude polysaccharides were obtained by concentrating and freeze-drying the resultant solution. Purification was carried out using an anion exchange chromatography on a DEAE-52 (GE Healthcare Life Sciences, USA) column (ID 20 mm × 50 cm) as described by Han et al. (20 ) Four fractions (AAP1, AAP2, AAP3, and AAP4) were obtained. A Sephacryl S-400 (GE Healthcare Life Sciences, USA) column (ID 20 mm × 50 cm), was used for further purification and the final polysaccharide fraction was lyophilized for further study

The mouse macrophage cell lines RAW 264.7, human monocytic leukemia THP-1, and transitional cell carcinoma of the bladder cell line T24 were purchased from ATCC (Rockville, MD, USA). Specific monoclonal antibodies, including antibodies against iNOS, NLRP3, IκB-α, p-IκB, p-Iκκ-α/β, Cox2, p65, p-P65 and GAPDH, were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Secondary antibodies for western blotting and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (Sigma Aldrich, St. Louis, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin solution were purchased from HyClone (Logan, UT, USA). Phycoerythrin (PE)-conjugated anti-CD40, CD16/32, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD282 and FITC-IgG2a antibodies were purchased from BD Systems (BD Biosciences, USA). ProcartaPlex™ Multiplex Immunoassay Kits were purchased from eBioscience (San Diego, CA, USA). DEAE-52 and Sephadex G-100 gel filtration medium was purchased from GE Health Care BioSciences AB (UPPPala, Sweden). SC75741 (purity, 99.79%) was purchased from Selleck (Shanghai, China).