Fig. 2.

Ubiquitination of AQP2 occurs before its internalization. MDCK cells expressing wt-AQP2 were pretreated with forskolin for 45 min (0). Subsequently, forskolin was washed out (forsk wash out) or the phorbol ester TPA was added (forsk + TPA) for the indicated times. Cells were lysed, and AQP2 was immunoprecipitated and immunoblotted for ubiquitin or AQP2. In parallel, forskolin-treated MDCK-AQP2 cells were surface-biotinylated in the cold, followed by an incubation at 37°C without forskolin (forsk wash out) or with forskolin and TPA (forsk + TPA) for the indicated times. Then, surface-bound biotin was removed with MesNa, and the cells were lysed. Biotinylated proteins were recovered by using immobilized streptavidin and immunoblotted for AQP2 (internalized AQP2).