Tissue protein extraction kit

The total protein of the mammary gland was harvested using a tissue protein extraction kit (Thermo Fisher Scientific, USA)^{17 (link)}. Ten percent or 15% SDS-PAGE was used to separate proteins, which were then bound to 0.45 μm PVDF membranes. After blocking in 5% skim milk, the prepared PVDF membranes were incubated at 4 °C overnight with specific primary antibodies, including p-p65 (#AF2006; 1:1000), p-65 (#AF5006; 1:1000), p-IκB (#AF2002; 1:1000), IκB (#AF5002; 1:1000), Occludin (#DF7504; 1:1000), ZO-1 (#AF5145; 1:1000), Claudin-3 (#AF0129; 1:1000), NLRP3 (#15101; 1:1000), ASC (#67824; 1:1000), IL-1β (#12242; 1:1000) and β-actin (#AF7018; 1:1000). After washing three times with TBST, the PVDF membranes were treated with goat anti-rabbit or rabbit anti-mouse IgG (1:20000) for 2 h at room temperature. Finally, the proteins were detected using the ECL plus western blotting Detection System (Tanon, China). All blots or gels derive from the same experiment and that were processed in parallel. Original blots are provided in Supplementary Information.

In order to detect the presence of pathogenic E. coli in the fecal samples, western blotting was carried out using anti-ETEC K88 and anti-ETEC K99 fimbrial antisera (SSI 51172, SSI 51173, VERITAS Co., Tokyo, Japan), and anti-ETEC 987P fimbrial antisera (originally generated in rabbit immunized purified pili of ETEC987P) for the determination of each pilus. Horseradish peroxidase conjugated anti-rabbit IgG was used as secondary antibody (7074, Cell signaling Technology Japan, K.K., Tokyo, Japan). All procedures followed to a commercial kit, ECL western Blotting Detection System (GE Health care Co., Tokyo, Japan). The feces samples were stirred vigorously by sonication and separated by centrifugation for 5 min at 20 °C. the precipitation was dissolved by using Thermo Scientific Tissue Protein Extraction kit (Tokyo, Japan), and purified by centrifugation. The supernatant was subjected for membrane blotting.