Anti p sapk jnk

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.