Fig. 3. PTEN overexpression and mutation regulate Akt phosphorylation on S473 and Akt activity. In A, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis using a P-S473-Akt antibody. In B, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by an in vitro kinase assay using GSK-3 as a substrate and Western analysis performed using an antibody to P-GSK-3. In C, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis for Akt. In D, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cell lysates are collected, followed by Western analysis for PTEN using a PTEN monoclonal antibody.