FIGURE 3.

Tests for a direct interaction between purified human Src kinase and purified human Na,K-ATPase. A, pull-down assay. The Na,K-ATPase (1.5 μg diluted 10-fold to reduce imidazole to 20 mm) was incubated with TEV-treated Src kinase at molar ratios of 1:2, 1:4, and 1:8 (Na,K-ATPase/Src) as described under “Methods.” Unbound, wash, and elution fractions were loaded on an SDS gel. Western blot was probed with anti-Src (n = 2). B, BN gel separation. Lanes 1 and 2, Coomassie stain. Lanes 3 and 4, Western blots probed with anti-Src. Lane 1, Src (8 μg). Lane 2, Src (8 μg) premixed with purified human Na,K-ATPase (α1β1FXYD1-NaK) (10 μg). Lane 3, Src (8 μg). Lane 4, Src plus Na,K-ATPase (α1β1FXYD1-NaK) (10 μg) (n = 4).