Figure 6

Ro5-4864 treatment attenuates activation of the PI3K pathway. (A) IgE-loaded BMMCs were pretreated for 20 min with 100 μM Ro5-4864, 100 μM clonazepam or the respective amount of DMSO and stimulated with Ag (DNP-HSA, 20 ng/ml) for the indicated times or left unstimulated. Subsequently, cellular lysates were analyzed by immunoblotting using antibodies against P-Akt (top panel), P-ERK1/2 (2^nd panel from top), P-IκBα (3^rd panel from top), P-p38 (4^th panel from top), and p85 (bottom panel, loading control). (B) IgE-loaded SHIP1+/+ and SHIP1−/− BMMCs were pretreated for 20 min with 100 μM Ro5-4864 or DMSO and stimulated for the indicated times with 20 ng/ml Ag. Cellular lysates were analyzed as described in (A). Comparable results were obtained with cells from different BMMC cultures. Densitometry was performed and relative expression levels are indicated under each band. (C) IgE-loaded SHIP1-deficient BMMCs were pretreated for 20 min with the indicated concentrations of Ro5-4864, clonazepam or a corresponding amount of DMSO and then either stimulated with 20 ng/ml Ag (DNP-HSA) or left unstimulated for another 20 min. Degranulation was measured by β-hexosaminidase assay. Each bar is the mean of duplicates ± SEM. Comparable results were obtained with cells from different cultures (n≥3). (D) IgE-loaded SHIP1-deficient BMMCs were pretreated for 20 min with the indicated concentrations of Ro5-4864, clonazepam or a corresponding amount of DMSO and then stimulated with 20 ng/ml Ag or left unstimulated for 3 h. IL-6 concentrations in the supernatants were determined by ELISA. Each bar is the mean of triplicates ± SEM. Comparable results were obtained with cells from different cultures (n≥3). Marks of significance (“asterisks”) relate to the respective vehicle (DMSO) control.