Fig. 7.

Effect of NHE-1 transfection on ouabain-stimulated Na-K-ATPase α1-subunit phosphorylation in HK-2 cells. Wild-type (WT) heme agglutinin (HA)-tagged NHE-1 was transfected into HK-2 cells as described previously (14). Twenty-four hours after transfection, cells were treated for 15 min with 10 pM ouabain. A: NHE-1 expression in vector- or NHE-1 WT-transfected cells was determined by immunoblotting using anti-NHE-1 antibodies. Bottom: expression of actin as control for protein loading. C: Na-K-ATPase α1-subunit was immunoprecipitated from crude membranes of vector- or NHE-1-transfected HK-2 cells treated with 10 pM ouabain and analyzed by immunoblotting using phosphotyrosine antibodies. Blots were stripped and reprobed using Na-K-ATPase α1-subunit antibodies. A representative immunoblot from 2 independent experiments is shown. B: NHE-1 WT- or vector-transfected HK-2 cells were treated for 15 min with 10 pM ouabain, and Na-K-ATPase-mediated ⁸⁶Rb uptake was measured as described in experimental procedures. Values are means ± SE from 6 independent experiments performed in triplicate. *P < 0.05 by 1-way ANOVA followed by Bonferroni analysis (GraphPad Prizm).