Effect of phosphorylation of RBC membranes by Syk and/or Lyn on band 3 binding to β-adducin in normal and ChAc RBC membranes. (A) Membranes (lanes 1 and 2) or Syk-phospho-membranes (lanes 3 and 4) of F2 RBCs from control (C) were incubated without (lanes 1 and 3) or with exogenous Lyn (lanes 2 and 4) and subjected to Western blot analysis with anti–phospho-Tyr (anti–pTyr) antibody (top panel) or were extracted with Triton X-100 and assayed after ultracentrifugation for the presence of band 3 by Western blot analysis (bottom panel). The membranes were reprobed with anti–actin antibody as loading control. (B) Band 3 was immunoprecipitated from membranes (lanes 1 and 2) and Syk-phospho-membranes (lanes 3 and 4) of F2 RBCs from C after incubation without (lanes 1 and 3) or with exogenous Lyn (lanes 2 and 4). The immunoprecipitates were subjected to Western blot analysis with anti–pTyr (top panel), anti–β-adducin antibody. The blots were also probed with anti–band 3 antibody. (C) Band 3 was immunoprecipitated from membranes of F2 ChAc RBCs previously incubated without or with the inhibitor PP2 (10μM). The immunoprecipitates were subjected to Western blot analysis with anti–β-adducin antibody. The blots were also probed with anti–band 3 antibody. The figure is representative of 3 independent experiments.