Recombinant il 1β

Mice were fasted for 6 h and given an i.p. injection of glucose (2 g/kg body weight; B. Braun, Germany). Blood glucose was measured at 0, 15, 30, 60, 90 and 120 min and blood was collected at 0, 15 and 30 min for insulin measurements. Recombinant IL-1β (1 µg/kg; R&D Systems, USA) or saline (154 mmol/l NaCl) was injected 18 min prior to the GTT, as described previously [25 (link)].

Recombinant IL-1β were purchased from R&D Systems (Minneapolis, MN). Antibodies for p-TAK1^(Thr184/187), TAK1^(Ser412), p-IRAK4^{(Thr345/Ser346)}, p-IRAK1^(Thr209), p-TAB2^(Ser372), IL-1β, p-STAT3^(Tyr705), p-STAT3^(Ser727), p-SAPK/JNK^{(Thr183/Tyr185)}, and IκBα were purchased from Cell Signaling Technologies (Danvers, MA; Cat# 90C7, 9339S, D6D7, T209, 8155, D3U3E, 9145S, 9136S, 4671S and 4812S). β-Actin and IRAK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; sc-47778; H273). Lipopolysaccharide (LPS), takinib and Tofacitinib were purchased from Sigma (St. Louis, MO, USA). The STAT3 Transcription Factor Assay Kit (Cat: 601950), 5Z-7-oxozeaenol, and NF-κB (p65) Transcription Factor Assay Kit (Cat: 10007889) were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and NG 25 trihydrochloride was purchased from Axon Medchem (Reston, VA, USA).

Human IEC: the small intestine enterocyte cell line CCL-241 and the colonocyte cell line CCL-248, human lymphoblastoid T-cell line CEM, and human monoblastoid tumor cell line U937 were purchased from ATCC (Manassas, VA) and grown in the respective ATCC complete growth media at 37°C in a humid, 5% CO₂ incubator. To differentiate into macrophages, the U937 cells were treated with 200 nM PMA (Sigma-Aldrich Corporation, St. Louis, MO) and allowed to adhere to tissue culture plate for 3 days [49 (link)]. The full length recombinant (r)SLURP-1 and rSLURP-2 were manufactured at Virusys Corporation (Sykesville, MD), as detailed elsewhere [50 ]. The previously characterized anti-SLURP-1 and -2 monoclonal antibodies 336H12-1A3 and 341F10-1F12, respectively [46 (link), 47 (link)], were from Research and Diagnostic Antibodies (North Las Vegas, NV). Normal mouse IgG (NIgG) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary mouse antibodies to human ICAM, IL-1β, IL-6, IL-10, TNFα, and IFNγ receptor (IFNγR) and ELISA kits for measuring human IL-6 and CXCL10 were purchased from R&D Systems (Minneapolis, MN). The IL-8 ELISA kit was from BD Biosciences (San Jose, CA). Both recombinant IL-1β and INFγ were from R&D Systems and both E. coli DNA and LPS from E. coli K12 strain (LPS-EK) were purchased from InvivoGen (San Diego, CA).

Adenosine-5’-triphosphate (ATP), N-acetyl cysteine (NAC), penicillin, streptomycin, HEPES, Mito-TEMPO, apocynin, carbenoxolone, paraformaldehyde, and bovine serum albumin were purchased from Sigma Aldrich (USA). Fetal bovine serum (FBS) was from Gibco/Life Technologies (USA). Z-YVAD and recombinant IL-1β were from R&D Systems (USA).

Bone marrow cells from TILRR^–/– and wild-type mice were cultured using standard conditions of 5% CO₂ at 37°C in 10% fetal calf serum Dulbecco’s modified Eagle’s medium supplemented with 20 ng/ml macrophage colony-stimulating factor for 7 days to stimulate differentiation into bone marrow–derived macrophages. A total of 2 × 10⁵ cells were seeded in 6-well plates and stimulated the following day with 1 nM recombinant IL-1β (R&D systems, Abingdon, United Kingdom) for required time. Cells were lysed in SDS lysis buffer (1% v/v Triton X, 0.5% [w/v] SDS and 0.1% dimethoxy-4-chloroamphetamine [w/v]). Lysates were vortexed and incubated on ice for 20 min, followed by 20 min centrifugation at 13,000 rpm (4°C).