Effect of increasing concentrations of α- and β- adducin tail domains on retention of band 3 in detergent-extracted membrane skeletons. (A) Increasing concentrations of α-adducin tail were incubated for 1 hour on ice with leaky ghosts (5 mg/mL protein). Mg²⁺ was then added to a final concentration of 4 mM, and the leaky ghosts were incubated for another 30 minutes. Ghosts were resealed in PBS for 45 minutes at 37°C and then extracted in 1% Triton X-100 (final concentration). Pelleted skeletons were analyzed by SDS-PAGE and immunoblotting using anti–band 3 and anti-actin antibodies. Densitometry of band 3/actin ratios revealed a reduction in band 3 content of 0%, 25%, 33%, and 37% in extracted membrane skeletons from red cells resealed with 0 μM, 3 μM, 9 μM, and 15 μM α-adducin tail, respectively. Whereas no change in spectrin, ankyrin, CD47, or glycophorin A retention was observed, protein 4.1, glycophorin C, and GAPDH content in the skeletons were reduced approximately 22%, 17%, and 31%, respectively. (B) Increasing concentrations of GST–β-adducin tail were incubated for 4 hours on ice with leaky erythrocytes (3 mg/mL protein) and then resealed for 45 minutes at 37°C. Resealed cells were extracted in 2% Triton X-100 (final concentration), and skeletons were analyzed by SDS-PAGE, followed by immunoblotting with anti-cdb3 and anti-actin antibodies. Densitometry of band 3/actin ratios indicate ∼ 50% ± 8% reduction in band 3 content at 15 μM GST–β-adducin tail, n = 2. Densitometry of band 3/actin ratios revealed a reduction in band 3 content of 0%, 17%, 21%, and 38% in extracted membrane skeletons from red cells resealed with 0 μM, 3 μM, 9 μM, and 15 μM β-adducin tail, respectively. Whereas no change in spectrin, ankyrin, CD47, or glycophorin A retention was observed, protein 4.1, glycophorin C, and GAPDH content in the skeletons was reduced ∼ 28%, 30%, and 25%, respectively.