P tbk1

Total protein extraction from mammary and colonic tissues and western blotting were performed according to previous studies [19 (link), 20 (link)]. In brief, mammary and colonic samples were weighed and homogenized using tissue protein extract (Thermo Fisher Scientific, USA). After centrifugation at 12,000×g and 4 °C for 10 min, the supernatants were harvested, and protein concentration determination was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). SDS-PAGE (12% or 15%) was performed to separate the targeted proteins according to molecular size. The targeted proteins were transferred to 0. 45-μm PVDF membranes and treated with 5% skim milk for 3 h at room temperature. Furthermore, specific primary antibodies, including p-p65, p-65, p-IκB, IκB, STING, p-TBK1, TBK1, p-IRF3, IRF3, occludin, ZO-1, and claudin-3 from Affinity Biosciences (OH, USA); TLR4, cGAS, NLRP3, ASC, and IL-1β from Cell Signaling Technology (Boston, USA); and β-actin from Immnoway Biotechnology (USA), were added for incubation at 4 °C overnight. After incubating with secondary antibodies (goat anti-rabbit IgG or rabbit anti-mouse IgG, Immnoway Biotechnology, USA) for 2 h at room temperature, the proteins were detected using an ECL plus western blotting detection system.

Antibodies for NF-κB p65 (#6956), p-NF-κB p65 (#3033) and β-Actin (#4970) (Cell Signaling Technology), p-TBK1 (#AF8190) and p-STAT3 (#AF3293) (Affinity Biosciences), STAT3 (#GB12176, Servicebio), HK2 (#22029-1-AP) and IRF3 (#11312-1-AP) (Proteintech Group), p-IRF3 (#MA5-14947, Invitrogen), TBK1 (#ab227182), Goat Anti-Rabbit IgG H&L (HRP) (#ab6721) and Rabbit Anti-Mouse IgG H&L(HRP) (#ab6728) (Abcam) were used for blotting. The anti-PDCoV-N antibodies were prepared in our laboratory. The Alexa Fluor 488-conjugated goat anti-mouse (#4408S) was purchased from Cell Signaling Technology. The STAT3 inhibitor static (#HY-13818) was purchased from Med Chem Express. The selenomethionine (SeMet) (#S3132) was purchased from Sigma. The miR-9-5p mimic, miR-125b-5p-1 mimic, and miR-125b-5p-1 inhibitor were purchased from Gene Pharma.