Fig. 8. Wrch-1 binds to the regulatory p85 subunit of PI3K and is necessary for Akt activation. (A) Wrch-1 depletion inhibits wound-induced Akt phosphorylation. Lysates were prepared as in Fig. 7A. Akt phosphorylation was visualized using an against antibody phosphorylated Akt (Ser473). An antibody against dynamin 2 was used to demonstrate equal loading. Levels of phosphorylated Akt from three independent experiments were quantified and normalized to levels of dynamin. ^*P<0.001, two-tailed t-test. (B) PI3K is necessary for cell migration. PI3K activity was inhibited by LY294002 (4 µM) (with overnight pre-treatment). Other conditions are as described in Fig. 5D. ^*P<0.0005 and ^**P<5x10^–6, two-tailed t-test. Data are representative of two independent experiments. (C) Wrch-1 binds to p85. Wrch-1 proteins were expressed in HelaS3 cells and pulled down using GST-p85 beads, as described in Materials and Methods.