FIGURE 8.

GDI-free pool of Rac molecules exists at the plasma membrane of resting cells. J774A.1 (A) and COS-7 (B) lysates were separated into membrane (M) and cytosol (C) fractions as described under “Experimental Procedures.” After SDS-PAGE, blots were probed for transferrin receptor (TfnR), β-tubulin, Rac, Rho, and RhoGDI. C, COS-7 cells were treated with luciferase- or RhoGDIα-specific small interfering RNA duplexes, as described under “Experimental Procedures.” After 48 h, cells were lysed and incubated with GST-YopO RBD or GST immobilized on glutathione-Sepharose beads. Bead eluates and samples of lysates were resolved by SDS-PAGE and Western blot, probing for RhoGDI (confirming knockdown), Rac, Rho, and actin (loading control). Data are representative of at least three independent experiments.