Figure 2: A) MCD4 cells were grown to confluence on glass coverslips and either left untreated (Ctr) or treated overnight (16 h) with 100 μM forskolin (FK) or with 5 μM fluvastatin (Flu). Cells were subjected to immunofluorescence with anti-AQP2 antibodies and analyzed in the xy plan by confocal microscopy.
B) MCD4 cells were grown to confluence into 150 cm2 cell culture Petri dishes and either left untreated (Ctr) or treated overnight (16 h) with 100 μM forskolin (FK) or with 5 μM fluvastatin (Flu). 25 ml of culture medium from each experimental condition were recovered and exosome isolated by ultracentrifugation. Exosomal proteins were separated by standard SDS-PAGE and analyzed by Western blotting with antibodies against AQP2 and flotillin-2 (Flot-2).
C) Densitometry of AQP2 bands was normalized for flotillin-2 abundance in each sample. Experiments were repeated three times with comparable results. * p<0.05; ** p<0.001.