Figure 4

Phosphorylation of eEF2 in response to IGF-I is PI3Kγ-dependent in MDA-MB-231 cells. (A) Spot map and three-dimensional view of differentially expressed proteins, the spot circled with pink line represents spot 101. (B). Graph view of spot 101. (C) Control or p110γ knockdown MDA-MB-231 cells were incubated in serum-free media for 1 hour before being stimulated with 0.1 nM IGF-I. Cell lysates were prepared and subjected to SDS-PAGE and Western blot to detect phosphorylated eEF2. Membranes were stripped and reprobed for eEF2 and β-actin as loading controls. These data are representative of at least 3 independent experiments conducted with similar results. (D) eEF2 phosphorylation was quantified by densitometry and normalized to the level of β-actin and expressed as a value relative to the 10 minute control-treated values (mean ± SEM of three independent experiments). * - significantly different from control values (2-way ANOVA with Bonferroni post-test) at *, p<0.05. (E) MDA-MB-231 cells were either untreated or treated with 2μM AS605240 and incubated in serum-free media for 1 hour and stimulated with 0.1 nM IGF-I. Cell lysates were prepared and subjected to SDS-PAGE and Western blot to detect phosphorylated eEF2. These data are representative of at least 3 independent experiments conducted with similar results. (F) eEF2 phosphorylation was quantified by densitometry, normalized to β-actin and expressed as a value relative to the 10 minute control-treated values (mean ± SEM of three independent experiments). * - significantly different from the control values (2-way ANOVA with Bonferroni post-test) at *, p<0.05.