Fig. 5

Analysis of the phosphorylation level of AQP2-WT, AQP2-R254W and AQP2-R254L in MDCK cells by means of Western blotting analysis. MDCK cells stably expressing AQP2 variant proteins were incubated with (150 μM) and without forskolin (+/-) for one hour. Following harvest and lysis, cell extract were subjected to SDS-PAGE and proteins were subsequently transferred to a PVDF membrane as described in Methods. Lanes 1, 2, 7 and 8: AQP2-WT; lanes 3, 4, 9 and 10: AQP2-R254W; lanes 5, 6, 11 and 12: AQP2-R254L. Following transfer, the membrane was cut in three separate pieces as indicated by the dotted lines and used for individual immunostaining analysis (a-c). (a) Loading control using an anti-ß-actin antibody. (b) AQP2 protein with S256 phosphorylation detected by a phosphor-sensitive anti-AQP2 antibody. The two asterisks denote p-AQP2-specific bands with an apparant higher mobility compared to the expected size of approximately 26 kDa. (c) Total amount of AQP2 protein as detected by an anti-AQP2 antibody. A molecular marker is indicated on the left (kDa). The presented Western blot represents the results obtained in two independent experiments