Polyclo nal rabbit anti occludin, anti claudin 1, anti claudin 3 and monoclonal mouse anti claudin 2 and antibodies were purchased from Zymed Laboratories. Horseradish Peroxidase anti rabbit IgG, HRP anti mouse IgG2b and Texas Red anti mouse IgG2b antibodies were purchased Lenalidomide price from Jackson Immu noResearch Laboratories, Inc. Alexa488 anti rabbit IgG antibody and Texas Red Phallodin were ERK1/2 Inhibition Cytokine induced Alterations in ERK1/2 Inhibition Reverses Cytokine induced Altera tions in Tight Junction Protein Distribution. Repre sentative immunoblots of occludin and claudin 1 using Triton X 100 soluble and insoluble fractions from confluent MDCK cell cultures treated for 24 hours in the indicated conditions. The effect of the MEK inhibitor was added fifteen minute prior to addition of proin flammatory cytokines.

Densitometic analyses were per formed, occludin and claudin 1, ratio of SDS to TX 100 intensity was reported. Error bars repre sent the mean SE of four independent experiments. ANOVA was performed, multiple comparisons between all treatments were determined with the Tukey HSD post test. Indicates statistically difference to the control group, indicates a significant difference to the TNF IFN group. Conclusion We demonstrated that MDCK tight junctions are func tionally reorganized in response to TNF IFN exposure through the activation of ERK1/2. We find the junction tightens by elevating the expression of occludin and clau din 1 in response to TNF IFN. Additionally, decreased ionic permeability arises primarily through a significant loss of claudin 2 expression due to ERK1/2 activation.

Apoptotic and necrotic mechanisms in response to TNF IFN may contribute in part to the elevated paracellular flux. Based on immunofluorescent findings, occludin and claudin 1 localization appear to be in transition perhaps purchased from Molecular Probes. A cyto toxicity kit was supplied by Roche Applied Science. D mannitol was purchased from Perkin Elmer and 4 KDa FITC dextran from Sigma Chemical. All other reagents were of the highest quality available. Cell culture MDCK cells were obtained from ATCC. MDCK cells were grown in Minimum Essential Medium Eagle supplemented with L glutamine, sodium pyruvate, non essential amino acids, 5% FBS, penicillin, streptomycin in a humidified incubator at 37 C and 5% CO2.

MDCK cells are passaged using a trypsin, EDTA solution and culture dishes are reseeded follow ing a 1 4 dilution. Laboratory grade water is used for all solutions and the water is routinely tested for the presence of endotoxin using the Limulus Amebocyte Lysate Assay. Cytotoxicity measurement Lactate AV-951 dehydrogenase activity released into the supernatant of MDCK cell cultures was used as a measure of cytotoxicity, manufacturers instructions were followed.