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. 2013 Dec 10;110(52):21124–21129. doi: 10.1073/pnas.1314124110

Fig. 2.

Fig. 2.

PI3K inhibition suppresses Rac but not RAS activation. (A) RAS activity assays were completed after 30 min of GDC-0941 treatment. Representative detection of active RAS (RAS-GTP, determined by the Raf-1 pull-down assay; Materials and Methods) is shown. The corresponding whole-cell extracts were probed with the indicated antibodies. Independent experiments were performed at least three times, and a representative result is shown. (B) Cells were transfected with control, K-RAS, or H-RAS–targeted siRNA for 72 h. Lysates were prepared and probed with the indicated antibodies. Results were confirmed by two independent experiments. (C) Cells were serum starved for 16 h, and then media containing 10% (vol/vol) FBS with or without 1 μM of GDC-0941 was added. Cells were lysed after 30 min, and Rac-GTP levels were determined with a PAK1-binding domain pull-down assay. Independent experiments were performed more than three times for T47D and twice for other cell lines, and a representative result is shown. (D) Cells were treated with 1 μM GDC-0941 for the indicated times, and lysates were probed with the indicated antibodies. Independent experiments were performed at least three times, and a representative result is shown. (E) Schematic representation of how PI3K is proposed to regulate ERK pathway.