Claudin 18

Specimens of cells and tissue were harvested and incubated for 10 min on ice with lysis buffer. The lysates were then centrifuged at 14,000 RCF for 10 min at 4 °C, and the supernatants were collected. Equal amounts of protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene difluoride membrane (Millipore). Western blot analyses were performed with the relevant antibodies: claudin-18 (diluted 1:200, Thermo Fisher Scientific Inc, IL, USA), p-SPAK (diluted 1:1000, OriGene, MD, USA), phosphorylated-p38 (p-p38) (diluted 1:1000, Cell Signaling Technology, USA), total-p38 (T-p38) (diluted 1:1000, Cell Signaling Technology, USA), beta-actin (diluted 1:1000, Sigma Chemical Company, MO, USA) and GAPDH (diluted 1:1000, Thermo Fisher Scientific Inc, IL, USA).

Specific antigen binding was performed by incubating sections with the respective primary antibodies for α-actinin (GeneTex, Irvine, CA, USA), desmin (GeneTex, Irvine, CA, USA), troponin I (Abcam, Cambridge, UK), connexin43 (Cell Signalling Technology, Cambridge, UK), zonula occludens-1 (Bioss, Woburn, MA, USA), claudin-18 (ThermoFisher, Waltham, MA, USA), and occludin (Bioss, Woburn, MA, USA) overnight at 4°C. Specific antibody binding was detected by using either AlexaFluor488-labelled (Jackson ImmunoResearch, West Grove, PA, USA), rhodamine-labelled (Jackson ImmunoResearch, West Grove, PA, USA), or AlexaFluor647-labelled (Jackson ImmunoResearch, West Grove, PA, USA) secondary antibodies. Cell nuclei were counterstained with Hoechst. Sections were analysed by fluorescence microscopy by using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Fluorescence intensities as well as the amount of apoptotic cells were evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Results are presented as mean fluorescence intensity.