Western Blot Analysis of Colon Tissue

Protein was extracted from the proximal 1 cm portion of the colon tissue using RIPA lysis buffer containing protease inhibitor and quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Then, 20 μg protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking for 1 h, the membranes were incubated with primary antibodies such as anti-Cytochrome C antibody (ab133504, abcam, Cambridge, UK); anti-Caspase 3 antibody (ab184787, abcam, Cambridge, UK); anti-Cleaved Caspase 3 antibody (ab214430, abcam, Cambridge, UK); anti-NLRP3 antibody (ab270449, abcam, Cambridge, UK); anti-Cleaved-IL-1ββ antibody (mAb#63124, CST, Boston, MA, USA); anti-phospho NF-kB p65 (Ser536) antibody (mAb#3033, CST, Boston, MA, USA); anti-phospho IκBα (Ser32) antibody (mAb#2859, CST, Boston, MA, USA); and anti-GAPDH antibody (AF0006, Beyotime, Shanghai, China) overnight at room temperature. Then, the membranes were incubated with the HRP-conjugated secondary antibody for 1 h. The color was developed with an electrochemiluminescence (ECL) reagent (Beyotime Biotechnology, Shanghai, China) and images were captured using the Bio-Rad gel imager (Bio-Rad, Hercules, CA, USA). The test method was based on previous papers from our research group [26 (link)].