Figure 5: Rab25 expression enhances EGF activation of AKT and MAPK pathways. (A) Western blotting analysis of EGFR, AKT and MAPK activation after EGF stimulation. Ovarian HEY Cells stable Rab25 expression and its control cells (pcDNA) were treated with 100ng/ml EGF for indicated time before protein isolation. Fold increase in phosphorylated protein (compared to 0 min EGF stimulation) after normalization of total protein was shown below the figure. (B) Decrease activation of AKT and MAPK signal transduction in MCF7 cells stable knockdown Rab25. (C) MAPK inhibitor PD98059 (10mM) but not by PI3K inhibitor PI103 (10mM) blocked OPG expression. a, p < 0.05 vs no stimulation control; b, p < 0.05 vs PD98059 alone. (D) Inhibition of MAPK signal transduction abolished EGF stimulation of OPG secretion in MCF7 cells. MCF7 shRNA control or Rab25 stably knockdown cells (shRab25) were pretreated with 10mM of PD98059, 1mM of AG1478 or 1mM of U0126 for 60min before addition of 100 ng/ml of EGF. a, p < 0.01 vs no stimulation control; b, p < 0.01 vs MCF7 sh Control. (E) Stimulation of OPG secretion by 100ng/ml of EGFR ligands. a, p < 0.01 vs no stimulation control; b, p < 0.01 vs MCF7 sh Control. (F) Inhibition of EGF stimulated OPG secretion by EGFR inhibitors. MCF7 cells were pre-treated with either 1mM of Lapatinib or Gefitinib for 60min before addition of 100ng/ml EGF. Media concentration was measured 24h posted addition of EGF. *, p < 0.05 vs no drug control.