FIGURE 1: Agonist-stimulated Akt Activation and PI3,4,5P₃ Generation at Internal Membranes.

a, b, c, Activation of Akt upon agonist stimulation. The activation of Akt in MDA-MB-231 cells at different time points following EGF or insulin or FBS stimulation were examined by immunoblotting using phospho-Akt antibody. Error bars denote mean±SD; n=4 independent experiments

d, Spatial localization of activated Akt and PI3,4,5P₃ upon EGF stimulation. MDA-MB-231 cells were fixed at different time points following EGF stimulation. Localization of activated Akt and PI3,4,5P₃ along microtubules was examined by immunostaining with antibodies specific to phospho-Akt and PI3,4,5P₃ respectively. The immunofluorescence signals of activated Akt and PI3,4,5P₃ at different time points were quantified. Similarly, the co-localization of activated Akt and PI3,4,5P₃ with tubulin were quantified by Pearson’s r. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

e, f Super-resolution microscopy revealing spatial localization of PI3,4,5P₃. MDA-MB-231 cells upon EGF stimulation were immuno-stained for PI3,4,5P₃ and tubulin and processed by STED microscopy. PI3,4,5P₃ signal was quantified in control vs. EGF stimulated cells. The relative level of PI3,4,5P₃ signal and co-localization with tubulin quantified. Scale bar, 1 μm; Error bars denote mean±SD; n=10 cells from representative experiments.

g, Quantification of activated Akt and PI3,4,5P₃ in plasma membrane and endosomes. MDA-MB-231 cells after EGF stimulation were harvested for subcellular fractionation. The activated Akt and PI3,4,5P₃ were quantified in the plasma membrane and endosomal fractions by immunoblotting and ELISA, respectively, as described in “Methods”. Error bars denote mean±SD; n=3 independent experiments

Unprocessed_Western_Blots_Fig1; Statistical_Source_Data_Fig1