Horseradish peroxidase conjugated anti mouse or rabbit igg

DMEM/F12 was purchased from Nacalai Tesque, Inc (Tokyo, Japan). RPMI 1640, foetal bovine serum (FBS), trypsin/ethylenediaminetetraacetic acid, H₂O₂, glycyrrhizin (GZA), glutathione (GSH) were obtained from Wako Pure Chemical (Osaka, Japan). Anti-Cx43 antibodies, 18α-glycyrrhetinic acid (α-GA), 18β-GA, lindane, flufenamic acid (FFA), carbenoxolone (CA), N-Acetyl-l-Cysteine (NAC), geneticin (G418) were purchased from Sigma-Aldrich (Tokyo, Japan). Antibodies against phospho-p38 MAPK (Thr180/Tyr182), β-tubulin, caspase-3 and horseradish peroxidase-conjugated anti-rabbit or mouse IgG were obtained from Cell Signaling Inc (Beverly, MA, USA). Antibody against TXNIP was purchased from MBL International (Woburn, MA, USA).

Proteins were extracted from the intestinal mucosa or cultured cells with complete radioimmunoprecipitation assay buffer and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (4–13% polyacrylamide)^{39 ,40} . The resolved proteins were then electrotransferred onto polyvinylidene fluoride membranes with a semi-dry blotter. The blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) with Tween 20 (TBS-T; 0.1% (v/v) Tween-20 in TBS, Sigma) for 1 h, washed with TBS-T, and incubated with a primary antibody at 4 °C overnight. The membranes were washed and incubated with a secondary antibody for 1 h. After washing, the membranes were incubated with chemiluminescent solution, and signals were detected. The primary antibodies used included rabbit polyclonal anti-5-HT₇ (#ab13898) (1:500, Abcam) and mouse monoclonal anti-β-actin (1:5000, Sigma). The secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or mouse IgG (1:1000, Cell Signaling).