Recombinant mouse ifn β

Mouse 129/Ola ESCs, NPCs differentiated in vitro from ESCs, and MEFs were cultured at 37°C with 5% CO₂ and routinely checked for the absence of mycoplasma contaminations as described previously (Bibel et al, 2007 (link); Teif et al, 2012 (link); Mallm et al, 2020 (link)). IFNβ was prepared from a BHK cell line over-expressing IFNβ and grown with DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. After growing the cells in the same medium but with 2% FCS for 24 h, the IFNβ containing medium was passed through a 0.45-µm sterile filter and stored in aliquots at −80°C. The activity of the resulting IFNβ stock was determined against a commercial preparation of recombinant mouse IFNβ (Sigma-Aldrich) by treating an Mx2-luciferase reporter cell line (IEC-Mx2Luc-10) for 24 h (Schwerk et al, 2013 (link)). A stock concentration of 16.6 U/µl was calculated, and the dose-response curve of the reporter signal in IEC-Mx2Luc-10 versus IFNβ concentration from Schwerk et al was reproduced. For the experiments described here, cells were treated with IFNβ at a concentration of 500 U/ml for 1 or 6 h. At this concentration, a strong albeit not saturated IFNβ response was achieved in the cell types studied.

Recombinant mouse IFN-β (Sigma-Aldrich, United States), antibodies against influenza virus proteins M1, NA, NP and PA (GeneTex, United States), antibodies against cellular proteins tubulin, p-STAT1, STAT1, p-TBK1 and TBK1 (Cell Signaling Technology, United States), horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology), DMEM, FBS, antibiotics (Hyclone, United States), lipopolysaccharide (LPS), bovine serum albumin (BSA) (Sigma-Aldrich), and 100 mm culture dishes and 6 or 96-well plates (Sarstedt, Germany) were purchased. Enzyme-linked immunosorbent assay (ELISA) antibody sets (eBioscience, United States), and RNA extraction kit (iNtRON Biotech, Korea), oligonucleotide primers for quantitative real-time polymerase chain reaction (qRT-PCR), DNA synthesizing kits, and the AccuPower® 2× Greenstar qPCR Master Mix (Bioneer, Korea) were procured.

Recombinant mouse IFN-β, ribavirin, zanamivir, and lipopolysaccharides from Escherichia coli were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-IRF3, anti-phospho-IRF3 anti-STAT1, anti-phospho-STAT1, anti-TBK1, and anti-phospho-TBK1 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA), and anti-β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against NS-1, PA, HA, PB-1, M2, and M1 were purchased from GeneTex (San Antonio, TX, USA).