Gapdh signal

Western blotting was used to detect the cleavage promotion of HN mutant proteins. As described previously, 1 μg of HN mutant plasmid and 1 μg of F plasmid were cotransfected into BHK21 cells. After adsorption at 37°C under 5% CO₂ for 36 h, the total protein was extracted from cells with radioimmunoprecipitation assay buffer (Solarbio, Beijing, China), separated by SDS-PAGE electrophoresis, and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The polyvinylidene fluoride membrane was blocked with 10% skim milk for 2 h at room temperature, and then a HA or Flag label-specific mouse primary antibody (1:3000; Invitrogen, Carlsbad, CA) was diluted with PBS and incubated with the membrane overnight at 4°C. The membrane was washed 5 times with PBS containing Tween-20 for 8 min intervals. The membrane was incubated with a secondary antibody (HRP-conjugated goat antimouse IgG, 1:3000; Invitrogen, Carlsbad, CA) for 2 h, and then the membrane was washed 5 times with PBS containing Tween-20 for 8 min each time. Finally, we used an ECL super sensitive kit (DiNing, Beijing, China) to expose the membrane by a chemiluminescence imager (MiniChemi610; Sagecreation, Beijing, China). The protein load was normalized to GAPDH signal (1:3000; Sungene Biotech, Tianjin Binhai New Area, China).