Western blotting for cell lines and isolated IECs was performed as described previously (Yulis, Quiros et al., 2018 (link)). In short, cells were lysed in RIPA (20 mM Tris-Base, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, pH 7.4) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and protein concentration was determined using Pierce Protein BCA kit according to manufacturer’s protocol. Samples were boiled for 10 min at 100°C for 10 min in NuPAGE LDS sample buffer (Life Technologies; Eugene, OR) with a final concentration of 100 mM DTT (Sigma-Aldrich) and 20 μg total protein was loaded onto polyacrylamide gels. After electrophoresis, the samples were transferred to a nitrocellulose membrane (Bio-Rad; Hercules, CA) and probed with primary antibodies diluted in 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween-20. Membranes were then incubated with appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies for 1 h at room temperature, followed by incubation with a chemiluminescence detection system (Clarity Western ECL Substrate). Finally, membranes were imaged by ChemiDoc imager (Bio-Rad).