9). of claudin-1, a major structural and functional Oxotremorine M iodide TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, Oxotremorine M iodide cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated. Introduction Enterotoxigenic (ETEC) represent an important cause of severe diarrhea in newborn animals [1] and diarrhea in humans following the ingestion of contaminated food and water [2]. Expression of both colonization factors and toxins are required for disruption of intestinal fluid homeostasis, leading to diarrhea [3]. ETEC strains are known to produce several types of enterotoxins, including heat-labile enterotoxin (LT), heat-stable enterotoxin a (STa) and heat-stable enterotoxin b (STb) [4]. Enteroaggregative heat-stable toxin 1 (EAST1) was also shown to be produced by ETEC [5], [6]. STb, a 48-amino-acid peptide of 5.2 kDa, secreted by ETEC strains is mainly associated with post-weaning diarrhea in piglets [7], [8]. producing a shiga toxin-independent non-bloody diarrhea [23], serotype O157:H7 [24], bacterial toxins such as toxins A and B [25], Zonula occludens toxin [26], and secreted autotransporter toxin [27] disrupt TJs [28]. Many studies on ETEC enterotoxins used T84 human colon cells, a cell line commonly used to study bacterial enterotoxin secretory processes [40]. Kreisberg et al. (2011) observed that LT-producing strains could affect cellular permeability independently of STa production [29]. However, in a study of Nakashima et al. (2013), STa elicited a reduction in TER and causes not only induction of water secretion but also intestinal barrier dysfunction but did not increase the paracellular permeability to FITC-labelled dextran [30]. For STb toxin, a reduction in TER associated with an increased in paracellular permeability was associated with a marked alteration of F-actin stress fibers [31]. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. Therefore, reduction in TER resistance and paracellular permeability to FITC-labeled dextran is recognized as indices of the decreased integrity of epithelial cells intoxicated with these Oxotremorine M iodide toxins. In a recent study, STb toxin generated an increase in cytoplasmically located TJ proteins including claudin-1 [31]. Less phosphorylated claudin-1 is found in the cytoplasm and highly phosphorylated claudin-1 is selectively concentrated at TJs monitored as NP-40-insoluble material [28], [32], [33]. Detergent insolubility of proteins is considered to indicate their integration into macromolecular phosphorylated complexes such as intercellular junctions [32], [34]. Membrane-associated claudin-l is known to be important structural and functional components in maintaining TJ integrity [35]. PKC, a family of serine-threonine kinases, are known to regulate epithelial barrier function. PKC are epithelial calcium-dependent enzymes and appears to regulate both subcellular localization and phosphorylation states of several TJ-associated proteins including claudin-1 [36]. The aim of the present study was to examine the effects of STb on location and phosphorylation state of claudin-1 in T84 intestinal epithelial cells. Materials and Methods Culture media, antibodies, and Thy1 reagents Dulbeccos modified Eagle medium (DMEM), Hams F-12 nutrient mixture (F-12), phosphate-buffered saline (PBS; pH 7.4, free of calcium chloride and magnesium chloride), 5% fetal bovine serum (FBS), rabbit polyclonal anti-claudin-1, goat anti-rabbit Alexa 488 antibodies, bovine serum albumin (BSA),.