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Fig. 3. Rac2^D57N expression in bone-marrow-derived macrophages inhibits M-CSF-stimulated surface ruffling. (A) M-CSF stimulates Rac activation in primary macrophages. Macrophages deprived of MCSF for 3 hours were stimulated for the indicated times with 100 ng ml^-1 of recombinant M-CSF. Cell lysates were incubated with GST-PAKcrib, and analysed with a monoclonal antibody to Rac. Rac activity was normalized to total Rac expression and data are expressed as the fold stimulation of Rac activation compared with basal activity. Representative blots from three experiments are shown. (B) F-Actin staining of M-CSF-starved and M-CSF-stimulated macrophages expressing Rac2^wt. F-actin was visualized with rhodamine-phalloidin. Scale bar, 10 µm. (C-E). Macrophages were transfected with empty vector, Rac2^wt or Rac2^D57N in pIRES2-EGFP treated as described above. Transfected cells were identified by EGFP expression. F-Actin was visualized with rhodamine-phalloidin. Quantitation of transfected cells with actin structures is shown. Data are expressed as mean±range of the percentage of transfected cells with actin structures from two independent experiments. A minimum of 50 transfected cells was examined for each condition.

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