FIGURE 7.

Unlike RhoGDI, YopO RBD preferentially binds Rac from cell lysates. J774A.1 (A) or COS-7 (B) lysates were incubated with GST-YopO RBD immobilized on glutathione-Sepharose beads, and bead eluates were resolved by SDS-PAGE and Western blotting, probing for Rac, RhoA, and β-tubulin. Results shown are representative of three experiments. C, after pulldown and SDS-PAGE, as described for A, bead eluates were stained by an MS-compatible silver nitrate stain. The 25-kDa band (boxed) was analyzed by MALDI-TOF MS. D, mouse Rac2 amino acid sequence, showing the overall sequence coverage of Rac1 and Rac2 peptides identified by MS (bold red). Amino acid differences between Rac1 and Rac2 are underlined.