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As described previously (Liao et al., 2019 (link)), cells transiently expressing the target proteins were fixed with 1.875% formaldehyde, permeabilized with 0.5% NP-40, and then incubated with anti-NS1 (sc-130568, Santa Cruz Biotechnology) antibody or anti-NP antibody (ab20343, Abcam) for 1 h. Cells were further incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen) for an additional 1 h at room temperature. Washing (PBS containing 1% FBS) was conducted between each step for a total of six washes. Subsequently, cells were treated with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) at a final concentration of 1 μg/ml for nucleic acid counterstaining. Images were acquired by confocal microscopy (FV1000, Olympus, Tokyo, Japan) with Olympus FV10-ASW 1.3 viewer software.

Samples of allantoic fluid from uninfected ECEs and ECEs infected with PR8-2344H5, PR8-2321H5, or PR8-H5-NS1(73)H5 virus were purified by ultracentrifugation through a 20% sucrose cushion. 293 T cells were transfected with plasmid pHW-NS1(73)-H5HA1-NEP as a control. Samples were mixed with Laemmli buffer containing β-mercaptoethanol and boiled for 5 min. Proteins were separated using SDS-PAGE, and targeted proteins were visualized by western blot using anti-H5 for clade 2.3.4.4 and clade 2.3.2.1 or anti-NS1 monoclonal antibody (sc-130568; Santa Cruz Biotechnology). Antisera were prepared by immunizing Balb/c mice with HA proteins from FJ/5 or JS/7 virus. For western blots, proteins were transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h in blocking buffer (150 mМ NaCl, 20 mM Tris–HCl, pH 7.5, 5% skimmed milk powder). Blots were probed with mouse anti-H5 serum at 1:2000 in blocking buffer containing 0.1% Tween-20 for 2 h at room temperature. Following three washes with TBST buffer (150 mM NaCl, 20 mM Tris–HCl, pH 7.5, 0.1% Tween-20), blots were incubated for 1–2 h at room temperature in HRP-conjugated goat-anti-mouse IgG antibody (Boster, Wuhan, China). The protein expressions were visualized by enhanced chemiluminescence system using SuperSignal West Pico (Thermo Fisher Scientific, MA, USA) (supplementary information).

For immunoblotting, MDCK cells infected with the viruses were lysed in RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, and 0.1% SDS). The proteins were then separated on 10% SDS polyacrylamide gels, then transferred onto a nitrocellulose membrane. The membrane was blocked in 5% skimmed milk buffer, then stained using primary monoclonal mouse antibodies against influenza A NS1 protein (1:2000; sc-130568; Santa Cruz, Dallas, TX, USA) and monoclonal mouse antibodies against influenza A virus NP protein (1:2000; sc-80481; Santa Cruz) at 4 °C overnight. For protein detection, the membrane was incubated with anti-mouse IgG HRP-conjugated secondary antibody and visualized using the ATTO Ez-Capture II system (ATTO, Japan). For the plaque assay, MDCK cells plated in 6-well tissue culture plates were inoculated with tenfold serially diluted viruses. After adsorption for 1 h, the cells were washed and overlaid with 1% low-melting agarose in DMEM containing 2% FBS. After incubation at 37 °C for 72 h, the agarose was gently removed and plaques were visualized with crystal violet staining.