Specific inhibition of band 3 secondary phosphorylation by either band 3 (1-26) phosphopeptide or recombinant Lyn SH2 domain.

The ability of Lyn to phosphorylate band 3 following phosphorylation by p36^syk with unlabeled ATP was measured in the presence of γ[³²P]ATP and increasing concentrations of either the indicated synthetic phosphopeptides and their unphosphorylated homologues (A) or recombinant GST and GST-SH2 domain (B). Incubations were carried out for 5 minutes under conditions described in “Materials and methods.” The samples were submitted to SDS-PAGE, and the radioactivity incorporated into band 3 was evaluated either by analysis on a Packard Instant Imager or by autoradiography and scintillation counting of the identified radiolabeled bands. Lyn activity is expressed as the percentage of the control values obtained in the absence of effectors. Reported values represent means of 4 separate experiments, with SE indicated by vertical bars.