The effect of CPZ on FK-mediated AQP2 phosphorylation and translocation. (A) AQP2-MDCK cells were pretreated with or without chlorpromazine (CPZ, 100 µM for 15 min), stimulated with forskolin (FK, 10 µM for 10 min), and then were subjected to Western blotting. The FK-induced increase of AQP2 phosphorylation at Ser-269 was not significantly changed by CPZ pretreatment. Protein band intensity was analyzed by ImageJ. Ser-269-phosphorylated AQP2 signals adjusted by total AQP2 signals were analyzed by a two-tailed Student t test (mean ± SD, n = 4, NS = not significant). The images are representative of three independent experiments. (B) AQP2-MDCK cells were pretreated with CPZ (100 µM for 15 min), then treated with FK for a further 10 min (right panel) or without FK (left panel), and then immunostained to localize AQP2. The larger panels represent confocal sections through the middle regions. The smaller horizontal strips at the bottom of each panel are Z-sections to show apical and basolateral membranes (taken in the plane indicated by the white arrows). AQP2 remained basolateral or intracellular, and FK-induced apical translocation of AQP2 was not observed after CPZ pretreatment. The images are representative of three independent experiments. Bar = 5 µm.