Hemagglutinin ha

The cultures were prepared similarly to the cultures prepared for the quantitative real-time PCR assay. However, after 24 h of incubation, the cells were washed with cold PBS and stored at −80 °C until use. The cells were then lysed with lysis buffer [50 mM Tris-HCl (pH 7.6), 120 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 50 mM NaF]. The protein concentrations of the lysates were determined using a protein assay kit (Bio-Rad Laboratories Inc., USA) and electrophoresed on 12% SDS-polyacrylamide gels [34 (link)]. The gels were electrotransferred to polyvinylidene fluoride membranes (PVDF 0.45 µm, Immobilon-P, USA). The membranes were blocked with a 5% skim milk solution (Becton Dickinson, USA) and then incubated with primary antibodies: neuraminidase (NA; Gene Tex, San Antonio, TX, USA), hemagglutinin (HA; Sigma, St Louis, MO, USA) or mouse monoclonal actin (Abcam, Cambridge, UK). After overnight incubation, the membranes were further incubated with secondary antibodies (Goat anti-Rabbit IgG (H + L) HRP or Goat anti-Mouse IgG (H + L) HRP; Thermo Sci., Rockford, IL, USA) for 2 h and detected by a chemiluminescence Western blotting detection kit (Thermo Sci., Rockford, IL, USA) using Image Reader LAS-4000 software.

Antibodies against Flag (catalog no. F1804) and hemagglutinin (HA) (catalog no. H6908) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against IFNAR1 (catalog no. ab45172) and NSE1 (catalog no. ab139689) were purchased from Abcam (Cambridge, MA, USA). Antibody against IRAK1 (catalog no. 4504S) was purchased from Cell Signaling Technology (CST) (Beverly, MA, USA). Antibodies against GAPDH (catalog no. 60004-1-Ig), GFP (catalog no. 66002-1-Ig), GST (catalog no. 66001-1-Ig), SMC5 (catalog no. 14178-1-AP), SMC6 (catalog no. 14465-1-AP), and PJA1 (catalog no. 17687-1-AP) were purchased from Proteintech Group (Wuhan, Hubei, China). Antibody against NSMCE4A (catalog no. AP9909a) was purchased from Abgent (San Diego, CA, USA).
Hepatitis B virus antigen detection ELISA kits were purchased from Ke Hua Bio Engineering (Shanghai, China). The inhibitors Braco19 and MG132 were purchased from Sigma-Aldrich. 3-Deazaneplanocin A (DZNeP), 5-aza-2ʹ-deoxycytidine (5′-Aza), GSK343, idarubicin HCl, and topotecan HCl were purchased from Selleck Chemicals (Houston, TX, USA).