Anti claudin 7

Total protein was extracted from CRC tissues and cultured cells and subjected to immunoblotting, as previously described [20 (link)]. Primary antibodies including rabbit polyclonal anti-claudin-1 (1:1000), anti-claudin-2 (1:500), anti-claudin-3 (1:1000), and anti-claudin-7 (1:1000) were obtained from Abcam (Cambridge, MA, USA). Primary antibody rabbit polyclonal anti-occludin (1:400) was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies including rabbit polyclonal anti-E-cadherin (1:1000), anti-c-kit (1:1000), anti-c-Jun (1:1000), anti-p-c-Jun (1:1000), anti-JNK (1:1000), and anti-p-JNK (1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-β-actin (1:2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The proteins were detected using enhanced chemiluminescence (ECL) (ThermoFisher Scientific, Waltham, MA, USA) and viewed in Fusion FX Vilber Lourmat (Paris, France).

Ileal and colonic samples were collected to measure the relative expressions of Claudin-7 and Occludin. Briefly, total proteins were extracted, and then the concentration of protein was determined by a bicinchoninic acid (BCA) kit (Pierce, Rockford, Illinois, USA). Next, the proteins were denatured, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to the polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk powder, followed by overnight incubation at 4°C with primary anti-β-tubulin (DSHB, Beijing, China), anti-Claudin-7 (Abcam, Nanjing, Jiangsu, China), and anti-Occludin (Abcam, Nanjing, Jiangsu, China) antibodies and appropriate secondary antibodies (CST, Nanjing, Jiangsu, China) for 2 h at room temperature. Finally, the bounds were visualized by the LI-COR Infrared Imaging System (Odyssey, Lincoln, NE).