Fig. 3.

Ouabain effects are mediated through Src in ADPKD cells. A: phosphorylation of Src. Cells were treated with or without 3 nM ouabain, either alone, or in the presence of tyrphostin AG1478 for 30 min. Lysates from the cells were subjected to immunoblot and phosphorylated Src was detected using an antibody against the activated Src (anti-Src-Y⁴¹⁸ antibody). Blots were then stripped and reimmunoblotted with an anti-total Src. Bars represent the relative phosphorylated amounts of Src, P-Src/Total Src, presented as a ratio of the untreated controls of 4 determinations performed on cells from 4 different ADPKD kidneys. Values different from the untreated controls are shown; *P < 0.001 and **P < 0.01. B: Src kinase activity. Cells treated in the absence and presence of ouabain were lysed and Src activity was measured using a method based on the incorporation of [γ-³²P] from [γ-³²P]-ATP into a Src-specific substrate peptide. Symbols represent data from 2 experiments and the means performed on cells from 2 different ADPKD kidneys. C: phosphorylation of Fyn. Cells were treated with or without 3 nM ouabain, lysed, and subjected to immunoprecipitation with anti-total Fyn antibody. After immunoblot, phosphorylated Fyn was detected using anti-phospho Fyn antibody. Total Fyn was determined in the imput material before immunoprecipitation. Bars represent the relative phosphorylated Fyn, P-Fyn/Total Fyn, presented as a ratio of the untreated controls.