Anti claudin 2

Colonic tissues were freshly isolated and embedded in paraffin wax after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on paraffin-embedded sections (4 μm), after preparation of the slides as described previously^{53 (link)} followed by incubation for 1 hour in blocking solution (2% bovine serum albumin, 1% goat serum in HBSS) to reduce nonspecific background. The tissue samples were incubated overnight with primary antibodies at 4°C. The following antibodies were used: anti-Claudin-2, anti-Claudin-7 (Invitrogen, Grand Island, NY, USA), anti-Salmonella Typhimurium (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Samples were then incubated with secondary antibodies (goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 488, Molecular Probes, CA; 1:200) for 1 hour at room temperature. Tissues were mounted with SlowFade Antifade Kit (Life technologies, s2828, Grand Island, NY, USA), followed by a coverslip, and the edges were sealed to prevent drying. Specimens were examined with a Zeiss laser scanning microscope (LSM 710 (Carl Zeiss Inc., Oberkochen, Germany).

Cultured cells were rinsed twice with ice-cold HBSS, lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and then sonicated. Mouse colonic epithelial cells were collected by scraping the tissue from the colon of the mouse, including the proximal and distal regions.^{51 (link), 52 (link)} 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. The following antibodies were used: anti-claudin-2, anti-claudin-3, anti-claudin-7 (Invitrogen, Carlsbad, CA, USA), anti-p-STAT3, anti-STAT3, anti-p-IκBα, anti-IκBα, anti-p-SAPK/JNK, anti-SAPK/JNK (Cell Signal, Beverly, MA, USA), anti-Villin and anti-VDR (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or anti-β-actin (Sigma-Aldrich, Milwaukee, WI, USA) antibodies and were visualized by ECL. Membranes that were probed with more than one antibody were stripped before re-probing.

Western blot analyses were carried out as described previously ^{39 (link)}. The following antibodies were used: anti-ZO-1, anti-occludin, anti-claudin-2 from Invitrogen; anti-MLC, anti-phospho-MLC and anti-IKKβ from Cell Signaling Technology (Beverly, MA); anti-TNF-α and anti-short MLCK from Sigma-Aldrich; anti-long MLCK from Abcam (Cambridge, MA) and anti-VDR from Santa Cruz Biotechnology.