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Fig. 10. SH3 domain of spectrin participates in Rac activation. (A) CHO cells expressing human {alpha}IIbß3 integrin were transiently transfected with HA-tagged SH3 domain of spectrin. Cells were serum-starved and plated on fibronectin-coated coverslips for 1 hour. Cells were fixed, permeabilized, and stained with antibody against HA epitope to detect transfected cells. Images are representative of four independent experiments. In all four experiments the transfection efficiency was ~30%. (B) CHO cells expressing human {alpha}IIbß3 integrin (control) or those that were also transiently transfected with HA-tagged SH3 domain, were serum-starved for 24 hours and plated on fibrinogen-coated dishes for 30 minutes, 1 hour or 3 hours. Cell lysates were incubated with GST-PBD beads, sedimented and bound Rac detected by western blotting. Lanes 1, 2 and 3 show bound Rac from lysates of cells expressing HA-tagged SH3 domain; lanes 4, 5 and 6 show bound Rac from control lysates. The bottom lanes show the amount of Rac in the total cell lysates. Data are representative of four independent experiments. Bar, 200 µm.