Electrophoretic pattern of ³²P-peptides obtained from tryptic digestion of band 3 phosphorylated either in vivo or in vitro by both p36^syk and Lyn.

(A) Intact erythrocytes were depleted of endogenous ATP and incubated with [³²P]-Pi as described in “Materials and methods.” Cell membranes were then rapidly isolated and submitted to SDS-PAGE. (B) Isolated erythrocyte membranes were phosphorylated with γ[³²P]ATP in basal medium containing 15-nmol/L p36^syk and 35-nmol/L Lyn and submitted to SDS-PAGE. In vivo and in vitro radiolabeled membrane proteins were transferred to polyvinylidine difluoride filters and treated with alkali to remove phosphoserine and phosphothreonine residues. ³²P-Y-band 3 was excised from the filters and digested with trypsin; resulting³²P-peptides were separated in 2 dimensions on thin-layer cellulose as detailed in “Materials and methods.” A similar amount of radioactivity was loaded on the cellulose plates. Shown are autoradiographs of the cellulose plates, with the origin indicated by an arrow. The figure is representative of 3 separate experiments.