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Fig. 6. Differential Triton-X-100 solubility of tight junction proteins and the effect of IFN{gamma} treatment on protein expression. (A) T84 cells were treated for 48 hours with 10 ng/ml IFN{gamma} and then extracted as described in Materials and Methods. Triton-soluble (S) and Triton-insoluble (I) fractions were immunoblotted with antibodies to claudin-1, claudin-2, claudin-3 and occludin. Images are representative of at least three separate experiments. As a positive control for claudin-2, the expression of this protein in untreated Caco-2 monolayers is also shown. (B) Densitometric analyses of immunoblots of claudin-1, occludin (65 kDa) and high molecular mass (phosphorylated) occludin in the insoluble fraction of control and IFN{gamma}-treated T84 monolayers. The graphs show the change in protein levels in IFN{gamma}-treated monolayers as a % of control and are mean data from five separate experiments. Significant differences in protein levels were observed; *P<0.05; **P<0.01 compared with controls as indicated by paired t-test.