Colonic Protein Expression Analysis

Colonic tissues or cells were lysed in Cell Lytic Mammalian Tissue Lysis Extraction Reagent (Sigma) containing the EDTA-free Protease Inhibitor Cocktail Set III, and the Phosphatase Inhibitor Set I (Millipore), and disrupted by sonication at 40 W. Protein concentrations were determined by the BCA Protein Assay Kit (Pierce). Total proteins (80 μg per lane) were separated on 10% polyacrylamide gels (Bio-Rad) and transferred onto PVDF membranes. Monoclonal anti-EspB (1:1000), rabbit monoclonal anti-Talin-1 Ab (1:2000), rabbit polyclonal anti-ACTN1 Ab (1:4000; Cell Signaling), rabbit polyclonal anti-SLC7A2 Ab (1:2000; Abcam), and mouse monoclonal anti-β-actin (1:10,000; Sigma) were used as described [9 (link), 10 (link)].