Detection of Tyr-phosphorylated band 3 in anti–SHP-2 IPs.

Red blood cells were treated in the absence (lane 1) or presence of 2 mmol/L diamide (lanes 2-4). Indicated concentrations of PP2 were added to the incubation mixture of assays of lanes 3 and 4. Erythrocyte lysates were immunoprecipitated with anti–SHP-2 antibody; immunocomplexes were subjected to SDS-PAGE and immunostained with anti–band 3 antibody (A). Blots were then stripped and reprobed with anti–P-Y antibody (B). In parallel experiments, blots were first immunostained with anti–band 3 antibody and then stripped and reprobed with anti–SHP-2 antibody (C). Panels are representative of at least 4 separate experiments.