Figure 3

Suppression of Akt by PTEN E307K mutant. (A) MDA-MB-453 cells were solubilized in 0.01% saponin-containing buffer. The membrane (Mem) and cytosolic (Cyt) fractions were resolved on SDS-PAGE gel and probed with an anti-PTEN monoclonal antibody (A2B1). The purity of the cytosolic and membrane fractions was confirmed with antibodies against tubulin and EGFR, respectively. (B) Pten ^-/- MEFS were transduced with the indicated retroviral expression plasmids. Cell lysates were prepared from cell lines and subjected to Western blotting analysis using the indicated antibodies. (C) MCF7 cells were electroporated with the indicated expression plasmids and total cell extracts were prepared for Western blotting analysis. Results from three independent experiments were quantified (right panel). Bars, SE. *, p < 0.05. (D) MDA-MB-453 cells were cultured in the presence or absence of 10% FBS. Cell extracted were prepared and the levels of p-Akt and c-Akt were monitored by Western blotting analysis (upper panel). MDA-MB-453 cells were treated with LY294002 (middle panel) or rapamycin (lower panel) at the indicated concentrations and durations. The activation states of Akt were monitored by Western blotting analysis with actin as a loading control.