Fig. 5. PTEN overexpression reduces basal levels of Akt phosphorylation at residues Ser473 and Thr308. (A) OBSCs derived from E14.5 CD1 embryos were infected with the PTEN-GFP or GFP construct and they were then stimulated with 100 ng/ml IGF-I or BSA (basal level) for 30 minutes under conditions of differentiation. Whereas IGF-I greatly stimulated P-Akt levels in both PTEN-GFP- and GFP-infected cells, the overexpression of PTEN reduced P-Akt basal levels. Similar results were obtained in samples from three experiments. (B) OBSCs derived from E14.5. Igf-I^–/– embryos were infected with the PTEN-GFP or GFP construct and were then stimulated with 10-100 ng/ml IGF-I or BSA (basal level) for 30 minutes under conditions of differentiation. The overexpression of PTEN reduced IGF-I-stimulated P-Akt levels only when 10 ng/ml of the growth factor was used. In B, to observe the signals corresponding to P-Akt basal levels, the films were developed for a long time, saturating for signals corresponding to IGF-I-stimulated P-Akt. For this reason, they are presented separately. Similar results were obtained in samples from two experiments.