Extended Data Fig. 1. Co-localization of Activated Akt and PI3,4,5P3 with Endosomal Compartments.

a, Spatial localization of activated Akt with endosomal vesicles. MDA-MB-231 cells stimulated with EGF were immunostained with antibodies for pAkt and clathrin (CHC) or early endosome antigen 1 (EEA1) or transferrin receptor (TFR). The co-localization of pAkt with CHC/EEA1/TFR was quantified in unstimulated vs EGF stimulated cells by Pearson’s correlation coefficient (Pearson’s r). Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments

b, c Abrogation of EGF stimulated Akt activation and PI3,4,5P3 generation by PI3K inhibitors. MDA-MB-231 cells were treated with wortmannin or LY294002 compound before EGF stimulation. Cells were harvested and processed for immunoblotting or immunostaining using antibodies specific for activated Akt or PI3,4,5P3. Activated Akt and PI3,4,5P3 levels were quantified. Scale bar, 5 μm; Error bars denote mean±SD; n=4 independent experiments (b), n=30 cells from representative experiments (c) </p/>d, e, f, Examination of the activation level of GFP-tagged full-length Akt1 and their localization upon EGF stimulation. Cos-7 cells transiently transfected with GFP-tagged full-length Akt1 were pre-treated with PI3K inhibitors before EGF stimulation. Localization of GFP-tagged full-length Akt1 with different endosomal vesicles were quantified. Scale bar, 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments </p/>g, Detection of PI3,4,5P3 in EGF stimulated cells by three different antibodies specific to PI3,4,5P3. Scale bar: 5 μm; Error bars denote mean±SD; n=30 cells from representative experiments Unprocessed_Western_Blots_Extended_Data_Fig1; Statistical_Source_Data_Extended_Data_Fig1