Ligation of CR1 promotes β-spectrin serine phosphorylation through a CK II-dependent mechanism. (A) RBCs were incubated with anti-CR1 mAb in the presence or absence of CK I or CK II inhibitors for 15 minutes at 4°C. RBCs were then washed and cross-linked for an additional 15 minutes with goat anti-mouse secondary Ab at 37°C. RBCs were washed, lysed, and tested for serine phosphorylation by immunoblotting with an anti-phospho-serine/-threonine Ab. Bottom panel shows β-actin as loading control. The experiments were done twice with similar results. (B) Inhibition of CK II blocks the effect of CR1 ligation on RBC membrane deformability. RBCs were incubated with anti-CR1 mAb followed by secondary cross-linking Ab in the presence of CK I or CKII inhibitors, and deformability was tested using microfluidic devices within 15 minutes. These results are representative of 2 independent experiments