Fig. 6. SWAP-70 binds to activated Rac. (A) Binding activity of SWAP-70 to Rho GTPases. Cell lysates from HEK-293T cells expressing GFP–SWAP-70 were incubated with GST-Rac loaded with GDP or GTPS, or GST-Rac free of guanine nucleotides (–), immobilized on glutathione-Sepharose beads. SWAP-70 trapped on the beads was analyzed by western blotting with anti-GFP antibody (upper panel). The filter was stained with Coomassie Blue to ensure that approximately equal amounts of GST proteins were present in each assay (bottom panel). A similar analysis was done using Cdc42 or Rho as probes. (B) Binding of SWAP-70 to Rac with mutations in the effector domain. Mutations shown in the figure were introduced into the effector domain of RacV12, a dominant-active form of Rac. The proteins were expressed as GST fusion proteins, and their binding activities to SWAP-70 were examined as described in A (upper panel). The amounts of GST fusion proteins used in the assay are shown in the bottom panel. Control, RacV12 without additional mutations.