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. 2002 Oct;22(20):7351–7363. doi: 10.1128/MCB.22.20.7351-7363.2002

FIG. 5.

FIG. 5.

Rai expression increases activation of PI3K/Akt by Ret. PC12 (A), PC12coRα (B), and SK-N-MC E/R (C) cells were infected with the empty vector (Ctrl) or with the vector containing Rai (Rai), serum starved, and treated with GDNF (100 ng/ml for results shown in panel A and 1 ng/ml for results shown in panel B) or EGF (100 ng/ml for results shown in panel C) at the indicated time points (0, 5, 15, 30, 60, or 120 min). Lysates were analyzed for activated Akt with antibodies specific for the phosphorylated Ser-473 residue of Akt (α-AktP) (top panels). Anti-Akt (α-Akt) and anti-vinculin (α-vinculin) antibodies were used to normalize the amount of proteins loaded. WB, Western blot; PDGF, platelet-derived growth factor. (D) In vitro kinase assay of anti-p85 immunoprecipitates from SK-N-MC E/R cells serum starved (SF) and stimulated with 100 ng of EGF/ml. Equal amounts of PI3K were confirmed by immunoblotting p85 in the cell lysates. PI3K activity is expressed in arbitrary units. The data in the graphs are presented as the ratios between the values obtained at each condition and those of the control cells (Ctrl) prior to EGF treatment (SF). The data represent the averages (± standard errors) of five independent experiments. Variations of PI3K activity that scored statistically significant (by Student's t test) with respect to the control cells are indicated with stars. In particular, for the control cells, the P values were 0.02 for 10 min of treatment and 0.007 for 60 min of treatment. For Rai-overexpressing cells, the P values were 0.09 for 10 min of treatment and 0.27 for 60 min of treatment. For Rai-overexpressing cells versus control cells, the P values were 0.009 for SF conditions, 0.019 for 10 min of treatment, and 0.0014 for 60 min of treatment. (E) Percent survival was measured for PC12coRα cells infected with either the Pinco retrovirus expressing Rai (Rai), the empty vector (Ctrl), or Rai FFF (Rai FFF), which had been maintained for 48 h in SF medium. Survival was determined by trypan blue exclusion. Bas, growing cells before starvation.