Phosphorylase inhibitor

High-efficiency RIPA lysis buffer supplemented with 1% protease inhibitor and 1% phosphorylase inhibitor (Beyotime, Shanghai, China) was adopted for total protein extraction from tissues and cells. Following protein concentration measurement using a BCA kit (Thermo Fisher Scientific), sample proteins were separated by 10% SDS-PAGE, transferred to a PVDF membrane, and then blocked with 5% BSA. Subsequently, the protein-loaded membrane was probed overnight at 4 °C with primary antibodies to UCHL3 (rabbit, 1:2000, ab241490, Abcam), AhR (mouse, 1:500, MA1-513, Thermo Fisher Scientific), PD-L1 (rabbit, 1:1000, ab205921, Abcam) and β-actin (rabbit, 1:5000, ab8227, Abcam; loading control), followed by 1.5-h incubation with HRP-labeled goat anti-rabbit IgG (1:20000, ab205718, Abcam) or HRP-labeled goat anti-mouse IgG (1:500, 31430, Thermo Fisher Scientific) at room temperature. The blots were visualized using developing solution (NCI4106, Pierce, Rockford, IL). Quantitative analysis was then conducted using ImageJ 1.48u software (Bio-Rad, HercμLes, CA).

RIPA lysate (P0013B Beyotime Biotechnology), PMSF (100 mM) (BL507A biosharp), Phosphorylase inhibitor (P1081 Beyotime Biotechnology), BCA protein quantitative detection kit (P0012 Beyotime Biotechnology), 5 × protein loading buffer (P0015 Beyotime Biotechnology), SDS–PAGE Gel preparation Kit (BL508A biosharp), Protein Marker (20350ES72 YEASEN), TRIS(1115GR500BIOFROXX), Glycine (1275KG2P5 BIOFROXX).

Total protein of tissue or cells was extracted from a high-efficiency RIPA lysis buffer (Sigma Aldrich) containing 1% protease inhibitor and 1% phosphorylase inhibitor (Beyotime, Shanghai, China). Concentration of the extracted protein was determined using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were separated by polyacrylamide gel electrophoresis, then transferred to PVDF membranes (Millipore, Billerica, MA), and blocked with 5% BSA for 1 h at room temperature. The membranes were incubated with rabbit anti-mouse primary antibodies against CTNNB1 (1:500, ab68183, Abcam) and GAPDH (1:500, ab8245, Abcam) at 4 °C overnight. The next day, the membranes were further incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:20,000, ab205718, Abcam) or goat anti-mouse IgG (1:20,000, ab197767, Abcam) diluent for 1.5 h at room temperature, followed by development using developing solution (NCI4106, Pierce, Rockford, IL, USA). Protein quantification analysis was performed by ImageJ 1.48 u software (2014, Bio-Rad Laboratories, Hercules, CA, USA) with an internal reference GAPDH.

DF1 cells were lysed with RIPA buffer supplemented with PMSF and phosphorylase inhibitors (Beyotime, Shanghai, China), and the protein concentration was tested with a Bradford assay (Bio-Rad, Hercules, CA, USA). Denatured protein (30 μg) was electrophoresed in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to an NC membrane (GE, MA, USA). NC membranes were blocked and incubated with specific rabbit anti-NLRP3 (provided by Professor Zhangyong Ning of South China Agricultural University), anti-NDV-NP antibody, or rabbit anti-GAPDH antibody (Cat: ab181602, Abcam, UK) for 12 h at 4 °C. The primary antibody was removed, and the membrane was incubated with IRDye 800-conjugated anti-rabbit IgG (Cat: 926-32211, LI-COR Biosciences, Lincoln, NE, USA) for 45 min at 25 °C–30 °C. The membranes were visualized using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).