r manuscript; available in PMC 2009 February 16. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript FIGURE 2. Effect of p110γ or p110δ ADX-47273 inhibition on adenosine-dependent Akt/PKB phosphorylation in mast cells and on adenosine-dependent vascular permeability. A , γKO and δD910A BMMCs were stimulated with adenosine or vehicle and Akt/PKB phosphorylation assessed by Western blotting, as described in Materials and Methods. A representative blot of two independent experiments is shown. Middle and right panels, BMMCs were pretreated for 30 min with varying concentrations of inhibitors, followed by adenosine stimulation for 1 min and immunoblotted for Akt/PKB.
GDC-0449 IC50 values were determined by ratiometric analysis of immunoblots, for which Akt/PKB phosphorylation was calculated as the ratio between phosphorylated Akt/PKB and total Akt/PKB for each lane and expressed as the percentage of Akt/PKB phosphorylation in the absence of inhibitor. A representative immunoblot of three independent experiments is shown. B, Impact of genetic inactivation of p110γ or p110δ on adenosine-induced PCA response in vivo. Number of mice used: WT and γKO, n _ 10 each; and δD910A, n _ 11. C, Impact of pharmacological inactivation of p110γ or p110δ on adenosine-induced PCA response in vivo. Number of WT mice dosed with AS605240, n _ 9 or IC87114, n _ 9. Ali et al. Page 12 J Immunol. Author manuscript; available in PMC 2009 February 16. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript FIGURE 3. Effect of p110γ or p110δ inhibition on SCF-dependent Akt/PKB phosphorylation and adhesion of mast cells.
A, γKO and δD910A BMMCs were stimulated with SCF or vehicle and Akt/PKB phosphorylation assessed by western blotting as described in Materials and Methods. A representative blot of two independent experiments is shown. , BMMCs were pretreated for 30 min with varying concentrations of inhibitors, followed by stimulation with SCF for 5 min and immunoblotted for Akt/PKB. IC50 values were determined by ratiometric analysis of immunoblots , as described in the legend to Fig.2. A representative immunoblot of three independent experiments is shown. B, Impact of genetic inactivation of PI3K isoforms on SCF-dependent mast cell adhesion. The experiment shown is representative of five independent experiments.
C, Impact of pharmacologic inactivation of PI3K isoforms on SCF-dependent mast cell adhesion. Graphs show data from a representative experiment done at least three or two times, with identical results. Ali et al. Page 13 J Immunol. Author manuscript; available in PMC 2009 February 16. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript FIGURE 4. Effect of p110γ or p110δ inhibition on IgE-dependent in vitro mast cell degranulation, Akt/ PKB phosphorylation, and PCA response in vivo. A, Effect of genetic inactivation of p110γ or p110δ on IgE/Ag-induced BMMC degranulation in vitro. Graph shows the mean _ SD of the following number of independent experiments: WT, n _ 10; δD910A, n _ 10; and γKO, n _ 8; done in quadruplicates. Mean _ SD spontaneous hexosaminidase release for experiments was as follows: WT, 8.
23% _ 1.83 ; δD910A, 11.88% _ 1.9 ; and γKO, 8.0% _ 1.4. B, Effect of isoform-selective PI3K inhibitors on IgE/Ag-induced BMMC degranulation in vitro. Graph shows data from two independent experiments _ SEM. C, Impact of PI3K inhibitors on IgE/Ag-induced Akt/PKB phosphorylation upon 30-s and 5-min stimulation of IgE-sensitized mast cells with Ag. A representative blot from three independent experiments is shown. D, PCA response of WT and gene-targeted mice. E, PCA responses of WT mice treated