Effect of impaired Rac activation on phagocytic target binding. (A) Detection of active Rac in untreated (DMSO) and in C. difficile toxin B– or NSC23766-treated cells. The presence of Rac-GTP was analyzed by pull-down experiments using PAK-PBD, followed by immunoblotting (top). Total Rac in an aliquot of the lysates was also probed by immunoblotting (bottom). The mean normalized ratio of PAK-PBD–bound Rac/total Rac from two independent experiments is indicated below the blot. (B) Quantitation of the effect of Rac antagonists on binding of IgG-opsonized beads. Data are presented as a binding index normalized to the vehicle control and are means ± SEM of three experiments, each counting at least 150 macrophages per condition. (C) Scanning electron micrograph of C. difficile toxin B–treated cells incubated with opsonized beads; bar, 5 µm. (D) Confocal microscopic images of RAW macrophages transfected with plasmids encoding either Rac1(T17N)-GFP or a plasma membrane–targeted GFP (PM-GFP). The transfectants were exposed to IgG-opsonized beads (blue) to assess binding efficiency; bar, 18 µm. (E) Mean binding index for cells transfected with either Rac1(T17N)-GFP, Cdc42(T17N)-GFP, or PM-GFP. Data were normalized to the binding observed in PM-GFP–transfected cells, used as a control, and are means ± SEM of three experiments each counting at least 60 cells.