Knockdown of PI3KAP/XB130 suppresses cAMP-dependent potentiation of IGF-I-induced DNA synthesis in FRTL-5 cells. FRTL-5 cells were transfected with control siRNA or siRNA against PI3KAP/XB130. After serum starvation, cells were pretreated with or without 1 mm Bt2cAMP for 24 h. A, The knockdown efficiency was evaluated by immunoblotting (IB). The means ± sem of the densitometric analysis results of three independent experiments are shown in the lower graphs. B, PI3KAP/XB130 tyrosine phosphorylation and its interaction with p85 PI3K were examined by immunoprecipitation (IP) 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. Similar results were obtained in two independent experiments. D–F, After washing to remove reagents, cells were treated with 100 ng/ml IGF-I for 24 h (D) or 3–6 h (E and F). [Methyl-3H]thymidine incorporation into DNA was measured during the last 4 h (D). The means ± sem of three replicate wells are shown. Similar results were obtained in three independent experiments. Cyclin D1 protein levels were analyzed by immunoblotting (E), and the cyclin D1 mRNA levels were analyzed by RT-PCR (F). RPS29 was a loading control. The means ± sem of the densitometric analysis results of three independent experiments are shown in the right graphs (E and F). *, P < 0.05. n.s., Not significant.