First, it corroborates the location the TED domain in the activated form of the bacterial protein

ere used and the degree of tightness of the TJ was measured by assessing the TER. Capsaicin Affects the Subcellular Lateral Localization of Factin Relative to TJ Proteins Reversible TJ Open by Cofilin-Actin and Occludin the lateral membrane, and claudin-1 localized along the lateral membrane and at the apical tip. This lateral membrane distribution of claudin-1 has been observed previously. In capsaicin-treated monolayers, F-actin concentrated to triTJ in XY sections and was closer to Zo-1, occludin and tricellulin in XZ sections. Therefore, capsaicin seems to induce the appearance of F-actin to a greater extent on the apical side than the basal side. This was continuous along the monolayers as seen in the 3-dimensional projection images. To confirm this phenomenon, the relative distributions of the proteins mentioned above and E-cadherin, an adherens junction Reversible TJ Open by Cofilin-Actin and Occludin protein localization. To our knowledge, this observed pattern is also capsaicin-specific in contrast to LatA and Jpk that affect TJ structure/organization. Occludin Protein Content and the Protein-protein Interactions Involving Occludin Reduced in TJ During the analysis of TJ HC-067047 components localization, we noticed the staining intensity of occludin diminished in capsaicin-treated monolayers. In fact, the intensity of occludin was markedly decreased compared to that of claudin-1 unchanged, suggesting that capsaicin may decrease the levels of occludin. Therefore, the levels of TJ proteins were assessed by western blotting of monolayers 21821695 with or without capsaicin treatment. The cytosolic and membrane fractions were analyzed separately because some TJ-altering treatments displace TJ proteins into the cytosol. In non-treated cells, occludin, tricellulin, claudin-1 and E-cadherin were mainly distributed in membrane fractions, and Zo-1 appeared in both fractions. Capsaicin 1685439 decreased the level of occludin in total cell extracts and membrane fractions without increasing the level in the cytosolic fraction, whereas the levels and distributions of claudin-1 and Ecadherin were unchanged. To determine whether the decreased occludin level and cofilin activation were capsaicin-specific phenomena, the effect of LatA, which decreases the TER irreversibly, on occludin and cofilin was assessed. Compared to capsaicin, LatA considerably decreased the TER at 60 min; however, occludin expression and cofilin phosphorylation were not affected. These data indicate that the decrease in occludin level and actin reorganization mediated by cofilin activation are capsaicin-specific mechanisms that reversibly increase TJ permeability. To assess the physiological/physical meaning of the decrease in the level of occludin, cosedimentation analysis was performed to investigate whether this decrease affects protein-protein interactions in TJ complexes. When monolayer lysates were subjected to velocity gradient centrifugation on sucrose gradients, part of occludin cosedimented with Zo-1, claudin-1 and tricellulin in fractions 1115, which is similar to the cosedimentation pattern of TJ protein complexes previously described. Their apparent molecular mass is greater than 200 kDa, which is higher than the monomeric mass of each. To confirm that these are not monomeric proteins, the monolayers were lysed with 1% SDS; occludin and Zo-1 concentrated in smaller molecular weight fractions as monomers. Taken together, figure 4D shows that the level of occludin, but not Zo-1 or claud