Fig. 4.

PI3KAP/XB130 is phosphorylated by Src family tyrosine kinases and forms a complex with PI3K. Quiescent FRTL-5 cells were treated with or without 1 mm Bt₂cAMP for 24 h. A and B, During cAMP treatment, the indicated concentrations of PP1 or PP2 were added. PI3KAP/XB130 protein levels, tyrosine phosphorylation, and its interaction with p85 PI3K were examined by immunoprecipitation (IP) and immunoblotting (IB) analysis. The means ± sem of the densitometric analysis results of three independent experiments are shown in the lower graphs (A). PI3K activity was measured in the immunoprecipitates with anti-PI3KAP/XB130 antibody. The means ± sem of three independent experiments are shown (B). C and D, Before serum starvation and cAMP treatment, FRTL-5 cells were transfected with control siRNA or siRNA against Src. PI3KAP/XB130 protein levels, tyrosine phosphorylation, and its interaction with p85 PI3K were examined by immunoprecipitation and immunoblotting analysis (C). PI3K activity was measured in the immunoprecipitates with anti-PI3KAP/XB130 antibody. The means ± sem of three replicate dishes are shown (D). E, The interaction of PI3KAP/XB130 with Src was analyzed by immunoprecipitation followed by immunoblotting with the indicated antibodies. Nonimmune IgG was used as a negative control. Similar results were obtained in two independent experiments. F, Src protein and the Y416 phosphorylation levels were analyzed by immunoblotting. The means ± sem of the densitometric analysis results of three independent experiments are shown as pSrc to total Src ratio in the graphs. *, P < 0.05.