Recombinant tnf α

Antibodies against β-actin (clone C4), NF-κB-p65 (clone C-20; C-terminus), NF-κB-p65 (clone F-6), and IκBα (clone H4) were purchased from Santa Cruz Biotechnology. Antibodies against NF-κB-p65 (clone C22B4; N-terminus), IKKα (clone3G12), IKKβ (D30C6), cleaved caspase-3 (Asp175, clone 5A1E), and PARP (clone 46D11) were obtained from Cell Signaling. SVA VP1 and SVA VP2 mouse monoclonals and SVA whole virus antibodies were kindly provided by Dr. Steve Lawson (SDSU). Anti-Flag or anti-HA mouse monoclonal antibodies are commercially available (GenScript and Thermo Scientific, respectively). Anti-rabbit and/or anti-mouse secondary antibodies conjugated with Alexa Fluor® 594 and Alexa Fluor 488® were purchased from Life Technologies. IRDye 800CW-labeled anti mouse IgG and IRDye 680-labeledRD anti rabbit IgG secondary antibodies were purchased from Li-Cor Biosciences. Recombinant TNF-α was obtained from InvivoGen. Staurosporine was purchased from Cell Signaling and Z-VAD-FMK was obtained from Santa Cruz Biotechnology.

THP1-Xblue-CD14 reporter cells (InvivoGen, France) were cultured and stimulated following manufacturer's instructions. Cells were incubated with 0.1–10 μM FYT21 or 1–40 μM HVF18 and stimulated with 1–1,000 ng ml⁻¹ of P. aeruginosa or E. coli LPS (Sigma), 1 μg ml⁻¹ purified LTA (InvivoGen), 10 μg ml⁻¹ zymosan A (Sigma), 100 ng ml⁻¹ purified flagellin (InvivoGen), 1–100 ng ml⁻¹ recombinant TNF-α (InvivoGen), 0.1% CM of various P. aeruginosa strains or no stimulus. In addition, cells and/or peptide were pretreated with 5–100 μg ml⁻¹ dermatan sulphate (DS36)41 (link). After 18–22 h incubation at 37 °C and 5% CO₂, 20 μl of the supernatants were transferred to new 96-wells plates and 180 μl of QUANTI-Blue (InvivoGen) was added. Plates were incubated at 37 °C and the levels of secreted embryonic alkaline phosphatase (SEAP), which is controlled by the activation of transcription factors NFκB and AP-1, were measured after 1–2 h at an absorbance of 600 nm. To establish the effect of FYT21 on activation of NFκB/AP-1 via TLR9, RAW-Blue reporter cells (InvivoGen) were cultured according to manufacturer's instructions, incubated with 10 μM FYT21 and stimulated with 500 ng ml⁻¹ ODN1826 (InvivoGen).