Fig. 1: NA and HA can be properly expressed from a single DNA construct by separation via furin cleavage sites and PTV-2A motif.

a Diagram of NA-F2A-HA expression vector. Open reading frame, ORF; furin protease, F; carboxypeptidase, CP; signal peptide peptidase, SPP. Created in BioRender. Leonard, R. (2024) BioRender.com/d14z470. b Cell-based ELISAs against 293T cells transfected with the indicated plasmids and stained with dilutions of an N2 polyclonal antibody. c Area under the curve-analysis (AUC) of 4 independent experiments represented in b. Arb. Units=Arbitrary Units. d Cell-based ELISAs against 293T cells transfected with the indicated plasmids and stained with an H3 HA-specific monoclonal antibody, 9H10. e AUC analysis of 4 independent experiments represented in d. f Confocal microscopy of cells transfected with the indicated plasmids, stained with Hoescht 33342 (blue), N2 polyclonal antibody (red), and H3 monoclonal antibody, 9H10 (green). g NA activity detected by ELLA assay using 293Ts transfected with plasmids expressing the indicated genes. h Confocal microscopy of HA fusion assay in 293Ts 24 h post transfection, stained with Hoescht 33342 (blue) and the fluorescent lectin, WGA (green). Panels b and d are representative of four independent experiments, and panels c, e, and g include means from all four independent experiments. For panels f and h, images were taken 24 h post transfection and are representative of two independent experiments. All scale bars = 30 μm. For all panels including statistics, significance was determined using Kruskal–Wallis test followed by pairwise Wilcoxon rank-sum tests with a Benjamini–Hochberg FDR correction. FDR-corrected p-values are reported above sample groups. Data are shown as means ± standard error of mean (SEM). Source data are provided as a Source Data file.