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Fig. 6. LPS stimulates Rac and p38 kinase activities in bone-marrow-derived macrophages and Rac2^D57N expression inhibits LPS stimulation of p38 kinase. (A) LPS stimulates Rac activation. Macrophages were stimulated for the indicated times with 10 ng ml^-1 LPS and cell lysates were incubated with GST-PAKcrib and analysed with a monoclonal antibody to Rac. Rac activity was normalized to the total Rac expression, and data are expressed as the fold stimulation of Rac activation compared with basal activity. A representative experiment of three such experiments is shown. (B) Measurements of the average nuclear phosphorylated p38 kinase intensity for either basal or LPS stimulation of macrophages is shown. Data are from seven independent experiments with duplicate coverslips. n indicates the number cells examined for each condition. (C) Immunoblot of lysates prepared from macrophages that were stimulated for 20 minutes with the indicated concentrations of LPS is shown. Blots were probed with an antibody that recognizes the phosphorylated form of p38 kinase. The figure shows a representative blot of two experiments. (D) Nuclear and phosphorylated p38 kinase immunostaining of untransfected macrophages unstimulated or stimulated for 20 minutes with 10 ng ml^-1 LPS is shown. Coverslips were treated as described in Materials and Methods. Nuclei were visualized with DAPI and phosphorylated p38 kinase was immunostained with an antibody to phosphorylated p38 kinase. A single section for each example is shown. (E) Data are expressed as the average nuclear phosphorylated p38 kinase intensity per nucleus of 15-25 transfected cells per treatment. Cells are from five independent experiments with duplicate coverslips. P values were calculated using Student's t test analysis. ^*, p38 kinase activity in Rac2^D57N-expressing cells stimulated with LPS was significantly different from both LPS-stimulated vector and Rac2^wt (P<0.01). ^**, Rac2^D57N basal expression was significantly different from both basal vector and Rac2^wt (P<0.005).

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