Irdye 800 or 680 secondary antibodies

HBEpCs were transfected with miR-4776 mimic and then mock infected or infected at a MOI of 1 IAV. Cells were also transfected with a negative control of mimic. Four hours post infection, the cells were lysed using a cytoplasmic protein extraction buffer containing protease inhibitors (Thermofisher Scientific, Foster City, CA, USA). The isolated proteins were electrophoresed on a 10% SDS-polyacrylamide gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Pre-cast gel, BioRad (Hercules, CA, USA). Separated proteins were transferred to a nitrocellulose membrane (Millipore) and the membrane was blocked using Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA). Western blot analysis was carried out using mouse monoclonal anti-phospho-NF-κB p65 (Millipore) and mouse monoclonal anti-GAPDH (Abcam, Cambridge, MA, USA) and NS1 of IAV (Invitrogen). Appropriate mouse and rabbit IR Dye 680 or 800 secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used. Near-infrared fluorescence detection was performed on the Odyssey Imaging System (LI-COR Biosciences).

HBEpC cells were transfected with DEFB1 plasmid or control plasmid for 48 h, cells were then exposed to A(H1N1) for an additional 24 h and protein extracts (30 µg) from the cells were prepared with 30 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) and analyzed by 10% SDS-PAGE. Separated proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked using Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) and probed with anti-STAT3, anti-phosphorylated-STAT3, and mouse monoclonal anti-GAPDH (Abcam) antibodies. Appropriate IRDye 680 or 800 secondary antibodies (LI-COR Biosciences) were used. Fluorescence detection was performed on the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and the signal intensities of the individual bands were determined. Protein quantitation was carried out using the Image lite software (LI-COR Biosciences).

HBEpCs were transfected with put-miR-34 mimic or inhibitor for 24 h and then mock infected or infected with H1N1 of MOI of 0.5 and 1.0. After 18 h post infection (p.i.), cells were lysed in 100 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail mixture (Thermo Fisher Scientific). Thirty micrograms of protein were solubilized in protein sample buffer and subjected to electrophoresis on 10% SDS-polyacrylamide gels (SDS-PAGE). Separated proteins were transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked using Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature and subjected to Western analysis with rabbit polyclonal anti-STAT3, anti-pSTAT3 (Abcam, Cambridge, MA, USA), and mouse monoclonal anti-GAPDH (Abcam, MA) antibodies. Appropriate mouse and rabbit IRDye 680 or 800 secondary antibodies (LI-COR Biosciences) were used. Near-infrared fluorescence detection was performed on the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and the fluorescent signal intensities of the individual bands were determined and normalized to GAPDH. All quantitation was done using the manufacturer’s software.

FMNL1 was detected using either a goat polyclonal antibody (Santa Cruz) or rabbit polyclonal antibody (Abcam). As a loading control, α-tubulin was detected using a mouse monoclonal antibody (B-1-5-2, Millipore Sigma). Antibody staining was visualized using the Odyssey near-infrared imaging system with IRDye-680 or-800 secondary antibodies (Li-cor Biosciences). Band intensities were quantified with densitometry using Odyssey 2.1 software (Li-cor Biosciences).