Il 1β

Anti-DNP IgE, DNP-bovine serum albumin (BSA), dexamethasone, and Evans blue were purchased from Sigma (St. Louis, MO, USA). The RBL-2H3 cell line (KCLB-22256) was purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and penicillin-streptomycin (PS) were purchased from Hyclone (Logan, UT, USA). The histamine, TNF-α, IL-6, IL-1β, and IL-4 ELISA kits were purchased from Biovision (Milpitas, CA, USA). Anti-phospho-p38 (Thr180/Tyr182), anti-phospho-ERK (L352), anti-phospho-JNK (T183/Y185), anti-p38, anti-ERK, anti-JNK, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Boehmeria nivea is an eco-friendly pesticide-free product, which was purchased from a farming association (Yeonggwang, Korea).

Fed hormones/metabolites levels were determined by collecting tail blood from mice that were without food for 3 h. Fasted hormones/metabolites levels were assessed in mice provided only with water ad libitum and without food for the indicated period. Time at day at which blood was collected was the same between groups. Tail vein blood was assayed for glucose levels using a standard glucometer (Nova Biomedical). Plasma was collected by centrifugation (1000 xg) in EDTA-coated tubes (Kent Scientific) and assayed using the indicated commercially available kits: β-hydroxybutyrate (Sigma), insulin (Crystal Chem. Inc.), TNF-α (Biovision), IL1-β (Biovision) and IL-6 (Biovision), S100A9 (R&D) and NEFA (Wako Chemicals).

LPS (Escherichia coli serotype 055:B5) was purchased from Sigma-Aldrich (L2880). To mimic the disease progression in human A-T, recombinant human TNFα (#1050) and IL1β (#4128) were chosen and purchased from BioVision. For use, each was dissolved in distilled water and stored at −20°C. Two treatment groups were established. In the first group, adult mice (2–3 months old) of either Atm^+/+ or Atm^−/− genotype were given daily intraperitoneal injections of LPS (1 mg/kg), TNFα (70 µg/kg), or IL1β (5 µg/kg) diluted in phosphate buffered saline solution, pH 7.4, for a period of 4 d. A control group was treated on the same schedule, but injected with filtered saline only. Mice were killed on the 5^th day, 24 h after the last injection. The second group of animals (Atm^+/+ and Atm^−/−genotype) was injected with LPS or filtered saline following the same schedule but was allowed to recover for 1 month after the last injection before being sacrificed for analysis. Mice in both groups were monitored carefully for signs of sickness or distress during the entire period. Following sacrifice, the brains were dissected and the tissues prepared as described below.

Cells or tissues were washed with cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors cocktail (Roche) followed by standard Western blotting procedure. After measuring protein concentration, the samples were loaded and separated on 10 to 12% precast polyacrylamide gels and then transferred to NC membranes (GE) at 100 V for 10 min. The membranes were blocked with 5% no-fat powdered milk and then incubated with primary antibodies overnight, followed by secondary antibodies. The specific signals were detected by Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) and the Tanon image system. The following antibodies were used: GAPDH (5174, Cell Signaling Technology), cGAS (31659, Cell Signaling Technology), STING (13647, Cell Signaling Technology), TBK1 (3504, Cell Signaling Technology), P-TBK1 (5483, Cell Signaling Technology), IRF3 (4302, Cell Signaling Technology), P-IRF3 (4947, Cell Signaling Technology), IL-1β (5129, Biovision), AKT (4691, Cell Signaling Technology), P-AKT S473 (4060, Cell Signaling Technology), and HRP-conjugated anti-rabbit IgG (H+L) as a secondary antibody (Beyotime).