Figure 5. AKT reactivation is Skp2-dependent.

(A) The indicated cell lines were infected with shRNA lentivirus targeting PIK3CA or control pLKO. Cells were serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+), harvested and lysates were immunoblotted with the indicated antibodies.

(B) MDA-MB-231cells infected with PIK3CA and/or SKP2 shRNA lentiviral vectors or control pLKO. SKP2 mRNA expression was measured by real-time RT-PCR and corresponding lysates were immunoblotted with indicated antibodies. Data are means ± S.E.M. from three independent experiments. ** P < 0.01 by a two-sided Student’s t-test.

(C) Immunblotting in lysates from MDA-MB-468 cells treated with BKM-120 (1 μM) for up to 144 hours, then treated again with BKM-120 (1 μM) for an additional 30 min.

(D) Immunblotting in lysates from MDA-MB-231cells infected with PIK3CA, SKP2, or PIK3CA in combination with SKP2 shRNA lentiviral vectors or control vector (pLKO) and serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+).

(E) Immunblotting in lysates from MDA-MB-468 cells infected with SKP2 shRNA lentiviral vector or control pLKO and then treated with BKM-120 (1 μM) for 3 or 48 hours.

(F) Immunblotting in lysates from MDA-MB-231 cells, parental and resistant to 1 μM BKM-120 (BKM-120), that were infected with SKP2 shRNA lentiviral vector or control pLKO vector and serum-starved overnight (−) or stimulated with IGF-1 for 20 min (+).

(G) MDA-MB-231 cells, parental and BKM-120–resistant, were infected with SKP2 shRNA lentiviral vector or control pLKO. After transfection with pcDNA3 or His-Ub, His-ubiquitin complexes were isolated, followed by immunoblotting. Blots are representative of at least 3 independent experiments. See also fig. S1D for additional time course with BKM-120 in cells expressing cDNA for SKP2.