The mem brane was then developed with ECL reagents and imaged with ChemiGenius Bio Imaging system. Optical density of protein signals were mea sured with ImageJ. Endogenous PINK1 was detected using Odyssey Infrared Imaging System. The epithelial barrier indicates the epithelial cell layer on the surface of mucosa such as airway and intestine. The epithelial barrier dysfunction is recognized in selleckchem a number of body disorders, such as intestinal allergy, inflammatory bowel diseases and asthma. The pathogenesis is un clear. Our previous studies reveal that microbial products, such as Staphylococcal enterotoxin B, can facilitate the development of immune disorders in the intestine. However, how the microbial products passing through the epithelial barrier to arrive the deep part of tissue is elusive.

The dysfunction of epithelial barrier manifests increases in the permeability to macromolecular molecules, such as protein antigens. The macromolecular substances may pass through the paracellular spaces, or to be transported via the transcellular pathway, to arrive the subepithelial re gion. Under healthy condition, epithelial cells endocytose some proteins of small molecular weight, those endocytic cargo can be wrapped by the plasma membranes to be formed as endosomes, the latter fuse with lysososmes where there are acidic enzymes to degrade the endocytic cargo. Recent reports indicate that there are a number of factors can affect the endolysosome systems to enhance the epithelial barrier permeability, the causative fac tors include microbial products, such as cholera toxin and SEB.

The underlying mechanism remains to be further understood. Alix Aip1 is a protein that functions in endosomal protein sorting, enveloped virus budding, and many other cellular processes. Crystal structures show that the Alix protein is composed of an N terminal Bro1 do main and a central domain, the latter consists of two ex tended three helix bundles that form elongated arms that fold back into a V. Alix binds to the endosomal sort ing complex required for transport to facilitate the membrane fusion events during the multivesicular en dosome formation. Based on the above information, we hypothesize that Alix is involved in the transcellular transport in epithelial cells. In this study, we observed that intestinal epithelial cell line, T84 cell, expresses Alix.

Ex posure to Brefeldin_A SEB suppressed the expression of Alix in T84 cells, which resulted in enhancing the epithelial barrier permeability to macromolecular antigens. Reagents The antibodies of Alix, TLR2, shRNA of TLR2 and shRNA of Alix were purchased from Santa Cruz Biotech. The reagents of qRT PCR, Western blotting and gene cloning were purchased from Invitrogen. SEB was purchased from Sigma Aldrich. The immune cell isolation kits were purchased from Miltenyi Biotech. The OVA ELISA kit was purchased from Antibodies Online. Mice The OVA TCR transgenic DO11.