Fig. 4.

Src is upstream of the PI3K/Akt pathway in metanephric mesenchymal cells. A: Src inhibitors PP2 and SU6656 inhibit PDGF BB-induced PI3K activity. Serum-deprived monolayers were pretreated with the indicated inhibitors and stimulated with PDGF BB. A PI3K assay was conducted as described in materials and methods. Thin-layer chromatography plates were activated in chambers containing saturation buffer and dried overnight. Immunoprecipitates of 100 μg protein were employed in a PI3K assay as described in the materials and methods. Reactions were separated by thin-layer chromatography. The arrow indicates phosphatidylinositol 3-phosphate. B: PI3K inhibitor LY294002 has minimal effect on Src phosphorylation in PDGF BB-treated metanephric mesenchymal cells. C: Src inhibitor PP2 abrogates PDGF BB-induced Akt phosphorylation in metanephric mesenchymal cells. D: adenoviral dominant negative (Ad DN) Src abrogates PDGF BB-induced Akt phosphorylation. Metanephric mesenchymal cells were infected with Ad DN Src or Ad green fluorescent protein (GFP) as a control. Quiescent cells were stimulated with PDGF BB, and immunoblotting was performed as described in materials and methods.