Band 3 Neapolis functions: binding of glycolytic enzymes and protein phosphorylation. (A) Aldolase binding to normal and mutant band 3. Red-cell membrane proteins from a healthy subject and the proband were separated by SDS-PAGE, transferred to a nitrocellulose paper, and analyzed by immunoblotting using an anti-aldolase antibody. (B) Sequential phosphorylation of band 3 in vitro. Red-cell membrane proteins were subjected to various treatments as outlined. Specifically, ghosts were incubated with Syk (lane 2) or with Lyn (lane 3) or without the enzymes (lane 1). Phosphate incorporation was evaluated on the basis of 32P signal (determined by exposing dried gels to x-ray films). In lanes 4, 5, and 6, membranes that were previously treated with Syk in the presence of unlabeled phosphate were incubated with Syk (lane 5) or with Lyn (lane 6) in the presence of labeled phosphate. Lane 4 is a control without addition of either Syk or Lyn. (C) Phosphorylation of band 3 in vivo. Red blood cells were incubated in the absence or presence of diamide. Cell membranes were isolated, solubilized, and submitted to SDS-PAGE followed by transfer to nitrocellulose. The upper part of blot shows immunostaining of band 3 with antiphosphotyrosine antibody. The filter was stripped and reprobed with anti-band 3 antibody (bottom panel). Nor indicates control membranes; Prob, proband membranes.
