Gb112182

Western blotting was performed as previously described [32 (link)]. The antibodies and dilutions used for Western blotting were as follows: anti-ALV-A envelope glycoprotein specific antibody (prepared in our laboratory, 1:1000), rabbit anti-TRAF6 antibody (bs-2830R, Bioss, China, 1:1000), rabbit anti-STAT3 antibody (bs-1141R, Bioss, China, 1:1000), rabbit anti-phospho-STAT3 (Ser727) antibody (bs-3429R, Bioss, China, 1:1000), rabbit anti-A20 antibody (GB112182, Servicebio, China, 1:1000), mouse anti-GAPDH monoclonal antibody (60004, Proteintech, USA, 1:5000), mouse anti-HA monoclonal antibody (M180, MBL, Japan, 1:3000), mouse anti-Myc monoclonal antibody (M047, MBL, Japan, 1:3000), mouse anti-Flag monoclonal antibody (Sangon Biotech, Shanghai, China, 1:5000), HRP-conjugated goat anti-rabbit IgG (Sangon Biotech, Shanghai, China, 1:5000), and HRP-conjugated goat anti-mouse IgG (Sangon Biotech, Shanghai, China, 1:5000).

The overexpressed adenovirus primers were designed according to NCBI sequences for chicken A20 (XR_005848478). A20 was amplified using PCR and cloned into the vector pShuttle-IRES-hrGFP-2. Two shRNAs were designed using an online website (http://rnaidesigner.thermofisher.com/rnaiexpress/) and cloned into the vector pShuttle-H1 after annealing. The primer sequences are listed in Table 1. The recombinant adenoviruses were constructed according to manufacturer's instructions (26 (link)). The constructed A20 adenovirus was inoculated into 10-day-old chicken embryos (10⁸ TCID₅₀/egg). The liver and kidneys were harvested 20 days after infection and treated with NP-40 cell lysate (P0013F, Beyotime, China). It was analyzed using Western blotting as previously described (27 (link)). The antibodies and dilutions used for Western blotting were as follows: anti-ALV-A envelope glycoprotein-specific antibody (prepared in our laboratory, 1:1,000), rabbit Anti-A20 antibody (GB112182, Servicebio, China, 1:1,000), mouse anti-GAPDH monoclonal antibody (60004, Proteintech, USA, 1:5,000), HRP-conjugated goat anti-rabbit IgG (D110058, Sangon Biotech, China, 1:5,000), and HRP-conjugated goat anti-mouse IgG (D110087, Sangon Biotech, China, 1:5,000).