Tissue protein extract

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.

Total protein samples were collected by a tissue protein extract (Thermo Fisher Scientific, USA), and protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific, USA). Targeting proteins were separated using 10% SDS-PAGE based on molecular size, and then proteins were bonded to 0.45-μm polyvinylidene fluoride (PVDF) membranes. After blocked in 5% skim milk for 3 h at room temperature, PVDF membranes were incubated with specific primary antibody (1:1000 for AhR, Cyp1a1, p-p65, p-IκB, p65, IκB, and β-actin) at 4°C overnight. Further, PVDF membranes were incubated with goat anti-rabbit IgG (1:20,000) for 2 h at room temperature after washing three times with TBST (Tris-buffered saline with Tween 20). Finally, proteins were tested using ECL plus the Western blotting detection system.

Western blotting of the total proteins extracted from placental or ileal tissues was conducted in line with a previous protocol [46 (link)]. In brief, placental or ileal tissues were homogenized with the tissue protein extract (Thermo Fisher Scientific, USA) and then centrifuged at 12,000 × g and 4 °C for 10 min to obtain the supernatants. The protein contents in these supernatants were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Targeted proteins were separated through 12% SDS-PAGE based on molecular size and then transferred onto the 0.45-μm PVDF membranes. Afterward, membranes were blocked using 5% defatted milk under ambient temperature for 3 h. Additionally, corresponding primary antibodies for antioxidant, apoptosis, ERS, autophagy, and voltage-dependent anion channel (VDAC) or β-actin were introduced for overnight incubation under 4 °C. Following 2-h incubation using goat anti-rabbit IgG secondary antibodies under ambient temperature, the ECL plus western blotting detection system was employed for protein detection. Protein expression was standardized to VDAC or β-actin expression and normalized to the mean ± SEM of the CON group. Table S3 presents more details about antibodies.