Pa5 349291

Vero E6 cells (2 × 10⁴) were cultured in 8-well chamber slides. The hCoV-OC43 and adapted-H1N1 co-infection and 3D8 scFv treatment were performed as described above. The slides were fixed with cold-methanol and permeabilized with an Intracellular Staining Perm Wash Buffer (Biolegend, USA) for 15 min each. Following blocking with PBS with 0.1% tween 20 containing 1% BSA and glycine for 1 h, the cells were incubated with polyclonal rabbit anti-HA antibody (PA5-349291, Invitrogen, USA), monoclonal anti-coronavirus antibody (OC43 strain, clone 541-8F, MAB9012, Sigma-Aldrich, USA), and anti-3D8 antibody (humanized antibody, clone 1D7, Bioneer, Republic of Korea) at 1:1000 dilution for 24 h at 4 °C. After that, goat anti-human IgG Alexa fluor 488 (A-11013, Invitrogen, USA), donkey anti-rabbit IgG Alexa fluor 555 (ab150074, Abcam, UK), and goat anti-mouse IgG Alexa fluor 647 (ab1500115, Abcam, UK) were incubated for 1 h at 25 °C. The nucleus was stained with VECTASHIELD Antifade mounting medium containing DAPI (LSbio, USA) and visualized using a Zeiss LSM 900 confocal microscope (Zeiss, German). The viral protein signals were converted to relative intensity percentages using CellProfiler 4.2.1, and the viral protein intensity was normalized to DAPI intensity.

Cells were lysed using RIPA buffer (Santa Cruz Biotechnology, USA) to extract the protein. Next, 20 μg of the protein was subjected to SDS-PAGE. Membranes after being transferred from gels were incubated with primary antibodies—monoclonal antibody to PEDV nucleoprotein protein (clone 3F12, 9191, Median Diagnostics, Republic of Korea), monoclonal antibody to hCoV-OC43 nucleoprotein (clone 542-7D, LS-C79764, LS-bio, USA), polyclonal rabbit anti-HA antibody to IAV (including H1N1 and H3N2) (PA5-349291, Invitrogen, USA), and polyclonal rabbit anti-GAPDH antibody (ab9485, Abcam, UK). After that, membranes were incubated with goat anti-mouse IgG-HRP conjugate (G-21040 Invitrogen, USA), and goat anti-rabbit IgG-HRP conjugate (A21020, Abbkine, USA). The membranes were added with Enhanced chemiluminescence (W3652-050, DawinBio, Republic of Korea) and exposed the film to observe the results. To analyze samples of viral co-infection, samples were divided into separated sets and then SDS-PAGEs were performed and transferred onto separated membranes. Each membrane was treated with different primary antibodies and secondary antibodies.