PI3Kα is required for EGF-stimulated PKB phosphorylation in MCF10a cells.

<p>MCF10a cells were serum-starved, pre-incubated with inhibitors or vehicle for 20 mins and stimulated with EGF (at the indicated doses) or its vehicle (the vehicle of the inhibitors was only was added to those samples stimulated with EGF without inhibitors or “starved”). After 15 mins the cells were lysed, aliquots were immuno-blotted with anti-β-COP (loading control, 110 kD), anti-phospho-T308-PKB and -S473-PKB antibodies simultaneously on the same filters. The immobilized antibodies were quantified with fluorescent 2° antibodies (goat-anti mouse-IRDye 800 for T308 and β-COP and goat-anti rabbit-IRDye 680 for S473 and a Li-Cor image analysis platform. Data are presented normalized to β-COP expression in the same sample. <b>Panel A</b>. shows a representative immuno-blot used to derive data shown in C and D. The final concentrations of the inhibitors with the cells were; A66, 6 µM; TGX221, 40 nM; IC87114, 1 µM; “mix”: A66, 6 µM+TGX221, 40 nM+IC87114, 1 µM; PI103, 1 µM. <b>Panel B</b>. The conditions of the experiments and the phosphorylation of S473-PKB was quantified, as in A (except, the experiment included PIK75 at 1 µM and the concentrations of A66, in µM, shown). The data are means ± SE (n = 3 experiments). The data indicate an IC50 of 800 nM. <b>Panel C</b>. The conditions of the experiments and the phosphorylation of S473-PKB was quantified, as in A. The data shown are means ± SE (n = 3 experiments). <b>Panel D</b>. The conditions of the experiments and the phosphorylation of T308-PKB was quantified, as in A. The data shown are means ± SE (n = 3 experiments).