Effect of diamide treatment on tyrosine phosphorylation and extractability of band 3 from erythrocyte membranes. Erythrocyte membrane proteins were separated by 8% SDS-PAGE and analyzed by Western blotting using anti-phosphotyrosine (anti-pTyr), anti–band 3, and anti-Syk antibodies. Gels were run under reducing conditions. (A) Erythrocytes were treated with different concentration of diamide (Dia) for 45 minutes at 37°C in the absence (lanes 1-5) or presence (lanes 6-10) of Syk inhibitors II and IV (Syk I). (B) 0.5% Triton X-100 (0°C for 15 minutes) was added to erythrocytes treated in the absence (lanes 6-10) or presence of Syk inhibitors (lanes 1-5), spun down, and the supernatant (Triton X-100 extract) was collected for Western blot analysis. (C) Membrane proteins and corresponding KI-IOVs (IOVs) obtained from control cells (lanes 1 and 3) and 2 mM diamide–treated cells (lanes 2 and 4). (D) Western blots of pelleted KI-IOVs obtained from control and diamide-treated erythrocytes after their incubation with (lanes 3 and 4) and without (lanes 1 and 2) ankyrin fragment (1nM). Control KI-IOVs (lanes 1 and 3) were obtained from the same experiment showed in Figure 2B. Images were acquired using a laser infrared fluorescence detector (Odyssey; LI-COR Biosciences).