Protein Extraction and Western Blot Analysis

Total protein was extracted by lysing cells in lysis buffer (20 mM HEPES, pH 7.4, 20 mM NaCl, 10% glycerol, and 1% Triton X-100). Colonic membrane proteins were prepared using a Membrane Protein Extraction Kit (Biovision, Milpitas, CA, USA) and soluble proteins were isolated from colon homogenates using methanol and chloroform (28 (link)). All protein samples were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Merck KGaA, Darmstadt, Germany). Immunoblotting was performed with the following primary antibodies: anti-tmTNF-α (home-made) (31 (link)), anti-TNF-α (Cat# 3707s), anti-PARP (Cat# 9532s), anti-cleaved caspase 3 (Asp175) (Cat# 9661s) from Cell Signaling Technology (Danvers, MA, USA), anti-IκB-α (Santa Cruz, CA, USA, Cat# sc-1643), anti-p65 (Cat# A19653), anti-p-p65 (Cat# AP0475), anti-caspase 3 (Cat# A17900), anti-Na⁺/K⁺ ATPase (Cat# A12405), and anti-β-actin (Cat# AC026) from Abclonal (Wuhan, China). HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA, Cat# 7074) were subsequently applied to the membrane. Bands were visualized using an enhanced chemiluminescence system (ECL; TIANGEN, Beijing, China).