Figure 2

ILK−IQGAP1-Ras complex sustains Ras activity to activate c-Raf/MEK1/2/ERK1/2/RSK/IκBα/NF-κB signaling. (A) Western blot of the indicated proteins in AGS and MKN45 cells. β-actin was used as an internal control. AGS cells treated with c-Raf inhibitor GW5074, MEK1/2 inhibitors U0126 or PD98059, or PI3K inhibitor LY294002 for 48 h were evaluated for their growth (B), phosphorylation of IκBα at Ser32 (pIκBα) and total protein (C), and NF-κB activation (D). (E) In shLuc- or shILK-transfected cells, western blots show the expression of the indicated proteins. β-actin was used as an internal control. (F) Ras activation assay showing Ras activity and protein expression in shLuc- and shILK-transfected AGS cells. (G) Representative fluorescence-based immunohistochemical staining showing the coexpression of ILK (green) and phosphorylated ERK1/2 Tyr202/Thr204 (red) in human gastric tumors and AGS-derived nodules in BALB/c mice. DAPI (blue) was used for nuclear counterstaining. The dot-plots show the coexpression of the indicated proteins. In control and IQGAP1 siRNA-transfected AGS cells at 48 h, the expression of the indicated proteins (H), NF-κB activation, and cell growth (I) were shown. Protein lysates extracted from untreated AGS cells (J) or with shLuc and shILK transfection (L, M) were immunoprecipitated (IP) with control IgG (C. IgG) or with antibodies against ILK, IQGAP1, and Ras. Immunoblots (IB) show the expression of ILK, IQGAP1, and Ras. (K) Representative fluorescence-based immunohistochemical staining showing the coexpression of ILK (green), IQGAP1 (red), and Ras (red) in AGS cells. DAPI (blue) was used for nuclear counterstaining. For proliferation and luciferase activity, data are mean ± SD from three independent experiments. **P <0.01 and ***P <0.001 compared with control. Upright triangle, increased expression; inverted triangle, decreased expression.