we observed a modest but not significant increase with all doses of TNF IFN tested. In order to examine dynamic changes in protein expres sion a differential detergent extraction procedure was employed using cytokine kinase inhibitor Sorafenib treated MDCK cells. Represent ative immunoblots of occludin and claudin 1 are pre sented in Figure 6A. The densitometric ratio of TX100 insoluble fraction to TX100 soluble fraction for occludin and claudin 1 was reported in Figure 6B. TNF IFN was added to the apical chamber, cells were incubated at 37 C for two hours, recovery of tracer was measured in the basolateral chamber and expressed as fold change from the control group. Error bars represent the SEM, n 3. A one way analysis of variance was performed, multiple comparisons between control and treat ments were determined with the Bonferroni post test.

Indicates statistical difference to TNF group. cantly lowered flux 60% back to control levels whereas SP600125 did not significantly alter flux. In these experi ments, inhibition of ERK1/2 and/or p38 signaling during TNF IFN exposure significantly protects MDCK cell bar rier function. Western blot analysis of tight junction related proteins To examine the causative factors related to elevated para cellular flux and decreased TER, selected tight junction gene products were examined by Western Blot. Occludin, 20 ng/ml exposure for 24 hours resulted in decreased occludin and claudin 1 in the TX100 insoluble fraction. MDCK cells pretreated with U0126 for 15 minutes prior to addition of TNF IFN showed a significant ele vation of occludin and claudin 1 in the TX100 insoluble fraction, this finding is consistent with the improved bar rier function in ERK inhibited TNF IFN treated MDCK cells.

These data suggest that early MAP kinase signaling events following TNF IFN exposure lead to both physi cal and functional remodeling of key tight junction pro teins. Tight junction localization studies Occludin, claudin 1, claudin 2 and claudin 3 localization was examined using indirect immunofluorescence in MDCK cells cultured on coverslips. Cells were treated for 24 hours in the absence and presence of TNF IFN. In this study, differences in expression and localization were quantified by examining junctional fluorescent intensity in a deconvoluted z stack comparing the highest intensity of staining to the adjacent intracellular location.

Occludin expression is robust and localized discreetly to the cells periphery. Examination of the effect of TNF IFN dose demonstrates an elevated occludin sig nal with a substantial increase detected at the cell cell Carfilzomib con tact regions. Analysis of occludin fluorescence intensity at the junction demonstrates a significant 55% increase in signal detected. Claudin 1 staining is more dynamic than occludin but generally the signal is local Discussion Epithelial cell layers play a critical role by separating phys iologically distinct compartments within most major organ systems.