Dephosphorylation of band 3 is inhibited by calpeptin in treated erythrocytes.

Erythrocytes were stimulated with 2 mM diamide in basal buffer containing calpeptin (2 mg/mL) for 20 minutes. In experiments of lanes 1-4, red cells were washed 3 times, resuspended, and incubated in basal buffer either in the absence (lanes 1, 2) or presence of calpeptin (lanes 3, 4) for 10 (lanes 1, 3) or 20 minutes (lanes 2, 4). Lane C refers to control erythrocytes treated with diamide and calpeptin without the following washes and incubation. Cell membranes were then isolated and solubilized, and an aliquot (15 μg) was submitted to SDS-PAGE, followed by transfer to nitrocellulose. Blots were immunostained with anti–P-Y antibody (A), stripped, and reprobed with anti–band 3 antibody (B). Panels are representative of 3 separate experiments.