Membrane ruffling and Rac activation in βig-h3–stimulated macrophages. Macrophages cultured on glass coverslips were either untreated (A) or stimulated with purified CD14 (B) or βig-h3 (C). The cells were fixed in formaldehyde and examined using a LSM510 laser-scanning microscope and Zeiss LSM Image Browser software. (D) Rac and cdc42 activation in βig-h3–stimulated macrophages. Macrophages were stimulated with βig-h3 or CD14 for 30 minutes or unstimulated before lysis. The cleared cell lysates were incubated with PAK-1 PBD-agarose. As a positive control, the unstimulated cell lysate was incubated with GTPγS before incubation with PAK-1 PBD-agarose. The bound proteins were subjected to Western blotting using anti-Rac and anti-cdc42 antibodies as indicated. As controls, an equal volume of total macrophage lysate for each treatment was also similarly analyzed. The blots were developed using alkaline phosphatase–conjugated goat anti–mouse IgG.