FIGURE 2.

Compound U18666A decreases plasma membrane cholesterol and specifically down-regulates α1 Na/K-ATPase. A, LLC-PK1 cells were treated with different doses of U18666A for 24 h and subjected to filipin (top row of upper panel) and α1 immunostaining (bottom row of upper panel). Representative images show dose-dependent effects of the U18666A compound on cellular distribution of cholesterol and α1 Na/K-ATPase. The intracellular and total α1 Na/K-ATPase signals were quantified by Image J software from randomly selected cells, and the ratio was calculated. Values are expressed as mean ± S.E. The number of the cells used for quantification was indicated as n value above the bar graph (bottom panel). **, p < 0.01 compared with control. Scale bar = 20 μm. U, U18666A. B, representative Western blot analysis from three independent experiments shows the effect of U18666A on the α1 subunit and α-tubulin after cells were treated for 48 h. C, representative Western blot analysis from three independent experiments shows the effect of U18666A on the protein levels of the α1 subunit, the insulin receptor β subunit, and α-tubulin after cells were treated by U18666A for 24 h and 48 h, respectively.