Figure 3.

In COS-7 cells incubated at 37°C, the effects of PDBu on the transport activity and the cell surface expression of Na, K-ATPase are independent of the phosphorylation of the α1 subunit at Ser-16. COS-7 cells stably expressing the wild-type (WT) Bufo α1 subunit, the T15A/S16A mutant, or the S16D mutant mimicking constitutive phosphorylation were incubated for 15 min at 37°C in the absence (C, open bars) or presence of 10⁻⁷ M PDBu (P, filled bars). (A) Exogenous Na,K-ATPase-mediated ⁸⁶Rb uptake was measured in the presence of 2.5 × 10⁻⁶ M ouabain under initial rates of influx. Results are expressed as picomoles of Rb (K) × minute⁻¹ × microgram of protein and are means ± SE from 7–12 independent experiments (*, p < 0.05; **, p < 0.01). (B) The effect of PDBu on the cell surface expression of Bufo α1 subunits was measured by Western blot with anti-Bufo α1 subunit antibody after streptavidin precipitation of the biotinylated cell surface proteins. Insets show a representative experiment, and bars show the quantitation of the relative amounts of cell surface Na,K-ATPase α1 subunits. Results are expressed as fractional change (with respect to control value) and are means ± SE from 6–18 independent experiments (*, p < 0.05).