Y-phosphorylation of band 3 in isolated ghosts and in intact human erythrocytes.

(A) Radioactive phosphorylation pattern of human erythrocyte membranes (ghosts) incubated with γ[³²P]ATP either alone (lane 1) or in the presence of 35-nmol/L Lyn (lane 2) or 15-nmol/L p36^syk (lanes 3-6). Increasing concentrations of PP1 were present in the incubation medium of assays shown in lanes 4 to 6. (B) Radioactive phosphorylation pattern of erythrocyte ghosts, first phosphorylated by p36^syk in the presence of unlabeled ATP as described in “Materials and methods” and further incubated for 10 minutes in the presence of γ[³²P]ATP without (lane 1) or with 35-nmol/L Lyn (lanes 2-5). Assays in lanes 3 to 5 were performed in the presence of increasing concentrations of PP1 added immediately before Lyn. ³²P-radiolabeled proteins were subjected to SDS-PAGE on 10% gels followed by autoradiography. (C) Anti-P-Y immunostaining of erythrocyte membrane proteins phosphorylated in vivo. Intact human erythrocytes were incubated either in the absence (lane 1) or presence (lanes 2-5) of pervanadate as described in “Materials and methods.” Increasing concentrations of PP1 were present in the assays shown in lanes 3 to 5. After incubation, erythrocyte membranes were rapidly isolated, solubilized, and submitted to SDS-PAGE followed by transfer to a nitrocellulose filter. The filter was then immunostained with anti-P-Y antibody. Other experimental details are described in “Materials and methods.” Panels are representative of at least 6 different experiments.