Anti claudin 4

To extract cellular protein, tissues were lysed by Pro-prep (catalog No. 17081; iNtRON Biotechnology, Korea), then homogenized by 20 strokes with a Dounce homogenizer, after which samples were centrifuged at 15,300 × g for 20 min at 4℃. Next, 50 µg of protein was loaded onto a 12.5% sodium dodecyl sulfate polyacrylamide gel, then transferred to nitrocellulose membrane (catalog No. IPVH00010; Millipore, USA). The blot was blocked with tris-buffered saline containing 0.5% tween-20 (TBS-T) including 5% skim milk for 1 h at room temperature (RT), then probed with the following primary antibodies at 4℃ overnight: anti-claudin-1 (1 : 1,000; 37-4900; Invitrogen, USA), anti-claudin-2 (1 : 1,000; 51-6100; Invitrogen), anti-claudin-4 (1 : 1,000; 36-4800; Invitrogen), anti-claudin-5 (1 : 1,000; 34-1600; Invitrogen), and anti-GAPDH (1 : 1,000; SC-137179; Santa Cruz Biotechnology, USA). After washing three times with TBS-T, the blots were treated with horseradish peroxidase-conjugated secondary antibody (bs-0296G-HRP; Bioss, USA; mouse IgG 1:3000 or sc-2004; Santa Cruz Biotech; rabbit IgG 1:3000) in TBS-T containing 5% skim milk for 2 h at RT. The blots were subsequently exposed to enhanced chemiluminescence reagent (SC-2048; Santa Cruz Biotech) and developed via GeneGnome5 (MOL5405; Syngene, USA). Signal specificity was confirmed through blotting without primary antibody.

Mouse colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions. The cells were sonicated in lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 8.0, 1% Triton X-100) with 0.2 mM sodium ortho-vanadate, and protease inhibitor cocktail. The protein concentration was measured using the BioRad Reagent (BioRad, Hercules, CA, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with primary antibodies. Villin was used as an internal control for intestinal epithelial cells. Beta-actin was also used as a control for equal loading of proteins. The following antibodies were used: anti-Claudin-10 (Invitrogen, 38-8400, Carlsbad, CA, USA), anti-Claudin-7 (Invitrogen, 34-9100, Carlsbad, CA, USA), anti-Claudin-3 (Invitrogen, 34-1700, Carlsbad, CA, USA), anti-Claudin-4 (Invitrogen, 36-4800, Carlsbad, CA, USA), anti-Claudin-7 (Invitrogen, 34-9100, Carlsbad, CA, USA), anti-villin (Santa Cruz Biotechnology, SC-58897, Dallas, TX, USA) or anti-β-actin (Sigma-Aldrich, A5316, St. Louis, MO, USA) antibodies and were visualized by ECL (Thermo Fisher Scientific, 32106, Waltham, MA, USA). Membranes that were probed with more than one antibody were stripped before re-probing.