FXYD3 gene silencing decreases the maximal Na,K-pump activity but not the cell surface expression of functional Na,K-ATPase. Maximal Na,K-ATPase activity was assessed by (A) ATPase activity measurements (4–7 independent experiments, *p < 0.034 vs. wild-type cells), (B) ouabain-sensitive 86Rb+ uptake (six independent experiments, *p < 0.018 vs. wild-type cells) and (C) short-circuit current measurements (3–5 independent experiments, *p < 0.049 vs. wild-type cells), 21 d after confluency of Caco-2 cells as described in Material and Methods. (D) [3H]ouabain binding to intact Caco-2 cells, 21 d after confluency (□). Nonspecific binding was determined in the presence of a 1000-fold excess of unlabeled ouabain (■).