Ligation of RBC CR1 induces serine/threonine phosphorylation of β-spectrin and adducin. (A) Cross-linking of RBC CR1 induces time-dependent serine/threonine phosphorylation, as assessed by FACS. RBCs were incubated with either control Ab or anti-CR1 for various amounts of time, fixed, permeabilized, and serine/threonine phosphorylation levels assessed by flow cytometry. (B) Western blotting analysis of the serine/threonine phosphorylation of β-spectrin (arrow) was temporally concordant with the FACS analysis. Control represents the basal serine/threonine phosphorylation levels of β-spectrin in fresh RBCs at 4°C. The experiment was repeated 4 times with similar results. (C) Phosphorylation levels of adducin increases 10 minutes after ligation of CR1. Control represents basal levels of phospho-adducin (lane 1), and as positive control, RBCs were activated by 0.5μM PMA (lane 3). Extra bands present in the anti-CR1 lane represent heavy and light IgG chains or the cross-linking anti-CR1 Ab. Loading controls are represented by the total amount of adducin in RBC lysate probed with rabbit polyclonal antibody anti-adducin. These results are representative of 3 independent experiments. (D) CR1-mediated adducin phosphorylation depends on Ca⁺⁺. RBCs were incubated with PMA or anti-CR1 in the presence or absence of Ca⁺⁺ (Mg⁺⁺ EGTA) and phosphorylation levels of adducin measured as above. Loading controls are represented by the levels of glyceraldehyde-3-phosphate dehydrogenase in RBC lysates.