Fig. 3. Perifosine decreases the levels of Akt and mTOR-associated proteins through ubiquitin/proteasome-mediated protein degradation.
A and B, the given cell lines were pretreated with 20 µM MG132 for 30 minutes prior to the addition of 10 µM perifosine (PRFS). After co-treatment for 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (A). In addition, the same lysates were also subjected to immunoprecipitation (IP) with rictor specific antibody followed by Western blotting (B). C, H460 cells were treated with 10 µM perifosine or PBS for 4 h. The cells were then washed with PBS twice and refed with fresh medium containing 20 µg/ml CHX. At the indicated times post CHX, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis.