FIG. 4.

Signaling response of wild-type and Gα15^−/− peritoneal macrophages to agonist stimulation and effect of pertussis toxin. (A-C) PI release in response to PAF, UTP, and C5a. Thioglycolate-elicited peritoneal macrophages from wild-type (wt) and Gα15^−/− mice were metabolically labeled with [³H]myo-inositol, and PI release was measured in 5-min time courses in response to PAF (30 ng/ml; A) UTP (10 μM; B), and C5a (100 nM; C). When indicated, cells were treated for 24 h with 100 ng of pertussis toxin (Ptx; islet-activating protein) per ml prior to stimulation with agonist. The data represents average ± standard error of the mean of a total of four experiments, three with pertussis toxin (A and B), and a total of seven experiments, four with pertussis toxin (C). The data are presented as counts per minute after subtraction of the background (bkg; counts per minute when cells are treated with buffer control). (D) ERK activation in response to stimulation with C5a. Macrophages were exposed to C5a (or buffer) for 3 min and lysed in buffer containing protease and phosphatase inhibitors; 20 mg of cell lysate was loaded per lane. When indicated, the cells were exposed to pertussis toxin (100 ng/ml) overnight prior to C5a stimulation. Western blot analysis was performed with antibody V6671 (Promega), which recognizes the dually phosphorylated ERKs. p44, ERK1; p42, ERK2; Ptx, pertussis toxin.