Identification of the adducin domain that interacts with KI-IOVs and band 3. (A) Native GST fusion constructs of intact adducin and its domains (1 μM) were incubated overnight at 4°C with KI-IOVs (65 μg in 200 μL; □) or with cdb3-(His)₆ (5.5 μM; ■). The KI-IOV suspension was pelleted through a 25% sucrose cushion, whereas the cdb3-(His)₆ solution was captured on nickel-nitrilotriacetic acid beads, washed 4 times, and eluted with 250 mM imidazole. GST activity was then quantified as a measure of adducin content. Data points represent mean ± SD, n = 2. Data were independently confirmed by dot blot analysis with anti-GST (data not shown). (B) Competitive inhibition of GST–β-adducin tail binding to KI-IOVs by intact erythrocyte adducin. KI-IOVs (100 μg of protein) were incubated with increasing amounts of intact erythrocyte adducin for 4 hours at 4°C (100 μL total volume), after which GST–β-adducin tail (250 nM) was added and incubated overnight at 4°C. Samples were processed and quantitated, as described above. Data points represent mean ± SD, n = 2 (apparent K_I ∼150 nM). (C) Verification of β-adducin tail binding with cdb3 by GST pull-down assay. GST-tagged cytoplasmic domain of band 3 was coupled to glutathione beads, pelleted, and washed (as described above). His-tagged C terminus of β-adducin was added to the mixture. The GST–band 3–conjugated beads (lane 1) at a final concentration of 1 μM were incubated for 1 hour at room temperature, pelleted, and then washed. The pellet was analyzed by SDS-PAGE, and β-adducin fragment was detected by Western blotting using anti-His antibody. GST-tagged cytoplasmic domain of glycophorin C (lane 2) and GST alone (lane 3) were used as negative controls. (D) Competitive inhibition of GST–β-adducin tail binding to KI-IOVs by anti-cdb3 antibody. KI-IOVs (70 μg, 200 μL total volume) were incubated with increasing amounts of anti-cdb3 antibody for 4 hours at 4°C, after which GST–β-adducin tail (1500 nM) was added and incubated overnight at 4°C. Samples were processed and GST activity was quantitated, as described above. Data points represent mean ± SD, n = 2. (E) The association of GST–β-adducin tail with band 3 requires Mg²⁺. His-tagged band 3 was immobilized on nickel beads and incubated with 270 nM GST–β-adducin tail in the presence of increasing concentrations of MgCl₂. Beads were pelleted, washed 5 times, and eluted with 250 mM imidazole in PBS. Eluted proteins were transferred to nitrocellulose membranes and probed for GST–β-adducin tail with an anti-GST polyclonal antibody. a.u. represents arbitrary units.