Effect of band 3 tyrosine phosphorylation on the ability of erythrocyte membrane KI-IOVs to bind ankyrin. Proteins were separated by 8% SDS-PAGE and analyzed by Western blotting using anti-phosphotyrosine (anti-pTyr) and anti-ankyrin antibodies. (A) Analyzed membrane proteins and KI-IOVs were obtained from control (lanes 1 and 3) and 2mM o-vanadate–treated (lanes 2 and 4) erythrocytes. (B) KI-IOVs obtained from control and o-vanadate–treated erythrocytes before (lanes 1 and 2) and after (lanes 3 and 4) incubation with the D3D4 domain of ankyrin (1nM). Quantification of ankyrin binding to KI-IOVs obtained from untreated (CTRL) and o-vanadate–treated erythrocytes in the absence (o-v) or presence of 6 μL of (400 units) λ phosphatase (o-v + Phosphatases) after incubation with different concentrations of the ankyrin fragment. (C) Quantitative densitometry of ankyrin was performed using a laser infrared fluorescence detector (Odyssey; LI-COR Biosciences) with Odyssey V3.0 software and is expressed as arbitrary fluorescence units.
