Figure 5.

A. Sucrose density gradient based fractions were prepared, as detailed in methods, and were probed for flotillin-1 as a marker for lipid rafts. The majority of flotilin-1 protein was present in low density fractions in control WT and ClC-2^−/− mouse colon (fractions 2–5). In DSS colitis, flotillin-1 protein content was shifted to high density detergent soluble fractions (fractions 6–10). B. In sucrose density gradient based fractionation studies, expression of occludin protein showed a significant shift from detergent insoluble fractions to detergent soluble fractions in DSS-treated ClC-2^−/− colon when compared to DSS-treated WT colon. C: The relative content of occludin in low density detergent insoluble (fractions 2–5) and high density detergent soluble (fractions 6–10) fractions was calculated by densitometry. #, *, p<0.05 vs. respective WT DSS. D: In similar sucrose density gradient studies, the presence of caveolin-1 within high density sucrose fractions in control WT and ClC-2^−/− colon was found to be shifted to medium density sucrose fractions after induction of DSS colitis. E: The densitometric values of occludin and caveolin-1, obtained from sucrose density fractions as in B and D, were used to calculate the ratio of occludin/caveolin-1 contents in respective sucrose fractions. In control WT and ClC-2^−/− colon, the occludin/caveolin-1 ratio showed a peak in the middle sucrose density fractions. Alternatively, the peak occludin/caveolin-1 ratio in DSS treated ClC-2^−/− was displaced toward high density, detergent soluble fractions, indicating higher content of occludin in association with caveolin-1 in these fractions (^*, different from respective fraction of WT DSS group, p< 0.01, representation of n=6).