0.45 μm polyvinylidene difluoride pvdf membrane

RAW 264.7 cells were treated with different concentrations of SCPs solution (300, 500, 750, and 1,000 μg/ml) for 48 h. The cell lysate was extracted on ice and centrifuged at 13,980 × g at 4°C for 20 min. The concentration of total protein was quantitated using a BCA protein assay kit (Beyotime, Shanghai, China). The cytosolic proteins were denatured by boiling in a loading buffer for denaturation. Equal amounts of protein (1 μg/μl) were loaded on the 10% SDS‐PAGE gel and transferred onto a 0.45‐μm polyvinylidene difluoride (PVDF) membrane (GE Healthcare, USA). Subsequently, the membrane was blocked using 5% non‐fat milk in TBST ((0.1% (v/v) Tween 20, 20 mM Tris‐HCl, 150 mM NaCl) for 1 h at 25°C, following incubation at 4°C overnight with the primary antibodies. These membranes were then washed three times (5 min/time) with TBST and incubated with the corresponding secondary antibody at 25°C for 1 h (Liu et al., 2019 (link); Rong et al., 2019 (link)). Immune complexes were visualized by a detection system using an enhanced chemiluminescence kit (Bio‐Rad).

Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin (P/S), Opti-MEM I (OMEM), anti-goat IgG HRP-linked secondary antibody, Simple Blue Stain, Novex 4–12% Tris-Glycine SDS-PAGE gels, dithiothreitol (DTT) Immulon-2HB 96-well plates, Nickel coated 96-well plates and Lipofectamine 2000 transfection reagent were all purchased from Thermo Fisher Scientific. Zanamivir and 2’-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) were acquired from Moravek Inc and Cayman Chemicals, respectively. β-propiolactone, formaldehyde, o-Phenylenediamine dihydrochloride (OPD), and anti-mouse IgG HRP linked secondary were purchased from Sigma. Anti-rabbit IgG HRP-linked secondary antibody and 0.45-μm polyvinylidene difluoride (PVDF) membrane were obtained from GE healthcare. Specific-Pathogen-Free (SPF) eggs and turkey red blood cells (TRBCs) were purchased from Charles River Labs and the Poultry Diagnostic and Research Center (Athens, GA), respectively. The H1N1 A/Brisbane/02/2018 field isolate (WT) and CVV (IVR-190) were kindly provided by the WHO. Rabbit Antisera against NA was generated by Agrisera (Sweden) using NA-WSN residues 35–453 isolated from E. coli inclusion bodies [29 (link)]. Polyclonal goat antiserum against the H1N1 influenza virus A/Fort Monmouth/1/1947 (NR-3117) was obtained from BEI Resources, NIAID, NIH.