FIGURE 10.

The block in HeLa cell transformation caused by the depletion of Cat-1 is rescued by knocking down Arf1. A and B, two sets of HeLa cells were transfected with the indicated combinations of Cont. siRNA, Cat-1 siRNA, and Arf1 siRNA. One set (A) was lysed and then immunoblotted with phospho-Akt (Ser-473), Akt, phospho-p70S6 kinase, and p70S6 kinase antibodies. Soft agar assays were performed (B) on the other set of cells. The number of colonies that formed for each condition was determined. C, HeLa cells transfected with the indicated combinations of Cont. siRNA, Cat-1 siRNA, and Pxn siRNA were lysed and then used in pulldown assays using the Golgi-associated, γ adaptin ear-containing, Arf-binding protein 3 (GGA3)-GST fusion protein prebound to beads. The cell lysates (Input) as well as the cell lysate proteins that bound the GGA3 fusion protein (GGA3 pulldown) were immunoblotted with an Arf1 antibody. All of the experiments shown were performed three separate times, each yielding similar results. The relative changes in Akt phosphorylation for each condition in A and Arf1 activation in C were determined. The data shown in B represent mean ± S.D. Student's t tests were performed. *, p < 0.05; **, p < 0.01.