FIGURE 9.

Paxillin inhibits Akt activity through an mTORC1-mediated negative feedback loop. A, HeLa cells transfected with the indicated combinations of Cont. siRNA, Cat-1 siRNA, and Pxn siRNA were lysed and then immunoblotted with phospho-Akt (Ser-473), Akt, phospho-p70S6 kinase (p-S6K), and p70S6 kinase (S6K) antibodies. B and C, two sets of HeLa cells were treated with the indicated combinations of Cont. siRNA, Cat-1 siRNA, rapamycin, and INK 128 (INK). One set (B) was lysed and then immunoblotted with phospho-Akt (Ser-473), Akt, phospho-p70S6 kinase, and p70S6 kinase antibodies. Soft agar assays were performed (C) on the other set of cells. The number of colonies that formed for each condition was determined. All of the experiments shown were performed three separate times, each yielding similar results. The relative changes in Akt and p70 S6 kinase phosphorylation for each condition in A and B was determined, whereas the data shown in C represent mean ± S.D. Student's t tests were performed. *, p < 0.05; **, p < 0.01.