Recombinant human saa

Murine macrophage-like RAW 264.7 cells were obtained from the American Type Culture Collection. Primary peritoneal macrophages were isolated from WT Balb/C, WT C57BL/6, or mutant C57BL/6 mice defective in either TLR4 or both TLR4 and RAGE (7 to 8 weeks, 20 to 25 g, male or female) at 3 days after intraperitoneal injection of 2 ml of thioglycolate broth (4%) as previously described (23 (link), 24 (link), 49 (link)). Human blood was purchased from the New York Blood Center (Long Island City, NY, USA), and human PBMCs were isolated by density gradient centrifugation through Ficoll (Ficoll-Paque PLUS) as previously described (23 (link), 24 (link), 49 (link)). Murine macrophages and human PBMCs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum or 10% human serum. When they reached 70 to 80% confluence, adherent cells were gently washed with, and immediately cultured in, Opti-MEM I before stimulating with crude LPS (E. coli 0111:B4; #L4130, Sigma-Aldrich), recombinant human SAA (catalog no. 300-13, PeproTech), HMGB1, or pCTS-L. The intracellular and extracellular concentrations of pCTS-L or various other cytokines/chemokines were determined by Western blotting analysis, cytokine antibody arrays, or enzyme-linked immunosorbent assay (ELISA) as previously described (23 (link), 24 (link), 49 (link)).

HAECs were purchased from Lonza (CC-2535; Tokyo, Japan) and used in all experiments. The HAECs were cultured in Endothelial Cell Growth Medium 2 (EBM-2) medium supplied with the EBM-2 bullet kit (Lonza, Tokyu, Japan) at 37°C with 5% CO₂. Subconfluent passage 6 cells were used in all experiments. HAECs were plated at 1.5 × 10⁵ cells/well in 6-well plates and cultured to subconfluence. The cells were then treated with 1.5 μg/mL recombinant human SAA (PeproTech, Rocky Hill, NJ) for 0, 1, 3 and 6 h.

Recombinant human SAA was purchased from Peprotech (Rocky Hills, NJ). The endotoxin levels were less than 0.1 ng/μg protein. Anti-β-actin antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, USA). Anti-NLRP-3 antibody was purchased from MERCK MILLIPORE (Billerica, MA, USA). Human IL-1β and caspase-1 (p20) ELISA kits were purchased from R&D systems (Minneapolis, USA). Anti-phospho-NF-κB p65 (Ser536) antibody was purchased from Cell Signaling Technology (CST, Danvers, USA). Hydroxychloroquine, iberiotoxin (IBTX), lipopolysaccharide (LPS), and adenosine 5 triphosphate (ATP) were purchased from Sigma-Aldrich (Tokyo, Japan).

Recombinant human SAA was purchased from Peprotech EC (Rocky Hill, NJ, USA). The functional grade anti-TLR2, anti-TLR4 and their isotype control IgG were purchased from Biolegend (San Diego, CA, USA) and the pam3CSK4 was bought from Invivogen (San Diego, CA, USA). Lipopolysaccharide (LPS; from Escherichia coli strain 055:B5), dexamethasone (DEX), Bay 11-7082, PD98059, LY294002, SB203580, oATP, polymyxin B (PMB), nonidet P-40 and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibody against phospho-ERK1/2, phospho-p38, and p38 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against ERK1/2, phospho-IκBα, IκBα, phosphor-p65, p65 and secondary antibodies were obtained from Santa Cruz (Dallas, TX, USA).

Recombinant human SAA was purchased from Peprotech (Rocky Hills, NJ). According to the manufacturer, the endotoxin level of the product is 0.1 ng/mg protein. Anti- IL-1β (pro-IL-1β, 3A6), anti-phosho-NF-κB p65 (Ser536) and anti-cleaved caspase-1 (D57A2) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-phosphotyrosine (clone 4G10), anti-Syk (clone4D10) and anti-caspase-1 antibodies were purchased from MERCK MILLIPORE (Billerica,MA USA). Anti-cleaved IL-1β polyclonal antibody was purchased from My BioSource (SanDiego, CA, USA). Caspase-1 inhibitor (Z-YVAD-FMK) was obtained from Abcam (Cambridge, UK). A Syk inhibitor, R406, was purchased from Selleckchem (Houston, Texas USA). Human IL-1β ELISA kit was purchased from R&D systems (Minneapolis, USA).