Western Blot Analysis of Colon Proteins

As previously described [23 (link),24 (link)], the RIPA protein lysate (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China) was employed to extract the total proteins from fresh colon tissue homogenates. The proteins were quantified with the bicinchoninic acid (BCA) protein assay kit (Biosharp, Wuhan, China) and denatured at 95 °C for 10 min, then separated by 12% SDS-PAGE gel electrophoresis, and transferred to the nitrocellulose membrane by electroblotting. The membrane was blocked with 5% skim milk, then incubated with primary antibodies against TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and cleaved Caspase 3, Bcl-2, Bax, NF-κB p65, p-STAT3 (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Proteintech, Wuhan, China) overnight at 4 °C. The HRP-labeled goat anti-rabbit or mouse secondary antibodies (Servicebio, Wuhan, China) were added to incubate for 1 h. The gray value of each strip was measured after ECL (Wuhan Kerui Biotechnology Co., Ltd., Wuhan, China) luminous development. β-actin was utilized as the control in the same sample.