Figure 5.

Mast cell but not macrophage activation is affected by FTI-277-mediated Ras inhibition. (A) DMSO- or FTI-277-treated WT-BMMCs were loaded overnight with anti-DNP IgE (100 ng/ml) followed by stimulation with DNP-HSA (Ag, 2 ng/ml) alone or in combination with 2µM Ade or 10 ng/ml SCF. Release of β-Hexosaminidase was quantified 20 min after stimulation (n = 6–7). (B) DMSO- or FTI-277-treated WT-BMMCs as well as p110γ^−/− BMMCs were exposed overnight to anti-DNP IgE (100 ng/ml) followed by stimulation with DNP-HSA (Ag, 2 ng/ml) together with 1 µM Ade for 6 h. TNF-α and IL-6 expressions were determined by qPCR and normalized to the level of GAPDH expression. Fold change of expression in FTI-277 treated or p110γ^−/− cells was quantified relative to DMSO-treated control (n = 6). (C, D) BMMCs and BMMØs were treated with either DMSO or 5 µM FTI-277 for 72 h, starved for 4 h, and activated with 2 µM Ade, 10 ng/ml SCF, 10 nM C5a, or 30 ng/ml M-CSF for 2 min at 37°C. Phosphorylation of PKB at Ser473 was determined by Western blot analysis of cell lysates with anti-PKB-Ser473 antibodies and normalized to the total level of PKB (n = 9–18). (E, F) Migration of BMMCs and BMMØs was assessed in Transwell chambers for 6 h at 37°C with indicated stimuli in the lower well, followed by quantification of migrated cells (n = 6–16). (E, F) were assessed with Student’s t-test and (B–D) were subjected to one-way ANOVA with Bonferroni’s post hoc test.