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Fig. 7. The correlation between claudin-7-GFP expression levels and changes in TER and dilution potentials. (A) Immunoblot analysis of claudin-7-GFP expression level. Three single clones were selected for each expression group. Lysates with equal total protein concentration were obtained from cells expressing low, medium and high levels of claudin-7-GFP protein. Immunoblots were probed with anti-GFP polyclonal antibody to detect claudin-7-GFP fusion protein (49 kDa) and GFP (27 kDa). Anti-actin staining was used as a loading control. (B) TER was measured, in the culture medium, on triplicate filters of three different clones expressing low, medium and high levels of claudin-7-GFP protein. A dose response increase in TER was correlated with the increasing expression level of claudin-7-GFP. (C) Dilution potential measurements were performed to determine the relationship between the ion selective and claudin-7-GFP expression level. Cells were grown on Snapwell filters for 8 days. Triplicate filters were used for measurement of dilution potentials in modified P buffer containing 140 mM lysine-HCl. When lysine-HCl concentration in the basal chamber was changed from 140 mM to 70 mM (column 1-4) or to 35 mM (column 5-8) while keeping the apical chamber constant at 140 mM, cells expressing low (column 2 and 6), medium (column 3 and 7) and high (column 4 and 8) levels of claudin-7-GFP displayed a progressive decrease in dilution potentials compared to that of control cells (column 1 and 5). (D) The experimental procedure was the same as described in C except that dilution potentials were measured in buffer containing sodium aspartate. Results indicated a dose response increase in dilution potentials from cells expressing increased levels of claudin-7-GFP (low: column 2 and 6; medium: column 3 and 7; high: column 4 and 8) compared to that of controls (column 1 and 5). *The value is significantly different from the control (P<0.05).





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