Fig. 4.

Ubiquitination of AQP2 increases its endocytosis. (A) MDCK cells expressing wt-AQP2, AQP2-K270R or AQP2-Ub were left untreated (−) or treated with forskolin, fixed, and subjected to immunocytochemistry using AQP2 antibodies. Confocal images were obtained in the XY and XZ planes. (B) MDCK cells expressing wt-AQP2 or AQP2-K270R were treated with forskolin, followed by TPA addition (forsk. + TPA) for the indicated times. Then, cells were fixed and analyzed as under A. (C) Internalization of wt-AQP2 or AQP2-K270R after forskolin treatment followed by TPA addition (forsk. + TPA) was determined as in Fig. 3 for the indicated times. Semiquantification of AQP2 (n = 3) was done as described in Materials and Methods. Filled and open data points represent wt-AQP2 and AQP2-K270R, respectively.