Figure 3

Phosphorylation of OPN induced Erk1/2 and c-Raf activation by Akt. (A) PC3 and PC3/OPN cells were immunoblotted with an Akt phospho-specific antibody (Akt-Ser473, upper panel). Membrane was stripped and blotted with an antibody to Akt to demonstrate total levels of AKT and equal loading (bottom panel). (B) PC3 and PC3/OPN cells were immunoblotted with an antibody to c-Raf Serine 259 (upper panel) and equal loading was shown using total levels of c-Raf (bottom panel). (C) PC3 cells were treated with either a vehicle or an Akt inhibitor overnight and immunoblotted with a phospho-specific antibody to c-Raf (Ser 259). (D and E) PC3 and PC3/OPN cell lines were pretreated with an Akt inhibitor for 16 h. The cell lysates were immunoblotted with phospho-specific antibodies to Erk1/2 (Thr202/204) or c-Raf (Ser338; upper panels in D and E). Membrane was stripped and immunoblotted with the corresponding non-phosphorylated antibodies to Erk1/2 and c-Raf to demonstrate equal protein loading (D and E-bottom panels). Results are representative of three independent experiments.