Figure 4

PI3K activation and decreased PTEN expression facilitate ERK1/2/NF-κB activation through ILK stabilization. (A) A PIP3 Mass ELISA Kit was used to determine PI3K activity by measuring the amount of PIP3 extracted from untreated AGS and MKN45 cells and from AGS cells treated with LY294002 for 24 h. (B) AGS cells were treated with PD98059, LY294002, T315, or Ras inhibitor FTI-277 for the indicated times. Western blots show the expression of the indicated proteins. β-actin was used as an internal control. (C) Ras activity was detected in AGS, MKN45, and LY294002-treated AGS cells after 24 h. Western blots show the expression of the indicated proteins in AGS cells pretreated with the translation inhibitor cycloheximide (CHX) (D) or proteasome inhibitor MG132 (E) for 0.5 h and then treated with LY294002 for 24 h. PTEN was overexpressed in AGS cells by using pcDNA3.1-GFP-PTEN (PTEN). Western blots show the expression of the indicated proteins compared with pcDNA3.1-GFP (Vector) transfection (F), and NF-κB activation (G) was also determined. For kinase and luciferase activity, data are mean ± SD from three independent experiments. *P <0.05, **P <0.01, and ***P <0.001 compared with control. Upright triangle, increased expression; inverted triangle, decreased expression.