Figure 4

Overexpression of KRAS, and differential activation of KRAS, p44/42 MAP kinase and AKT in KRAS -amplified gastric cancer cells. (a) Immunoblot analysis of the expression levels of KRAS and NRAS in cancer cells. Actin expression was analyzed as a loading control. (b) The basal level of GTP-KRAS was markedly high in gastric cancer cells with amplified mutant KRAS (HSC45 and SH101P4). Total lysate (500 μg) was subjected to a GTP-RAS pull-down assay, and GTP-KRAS and GTP-NRAS were detected by immunoblot using anti-KRAS and anti-NRAS antibodies, respectively. Total cell lysate (50 μg) was analyzed in parallel to determine the level of expression of KRAS and NRAS in cells. (c) GTP-KRAS was elevated after serum stimulation in MKN1 cells. Cells were cultured in regular medium containing 10% FCS (N), serum-starved for 24 h (-) or serum-starved then stimulated with 10% FCS for 1 h (+). Total cell lysate was subjected to a GTP-KRAS pull-down assay. (d) Activation of p44/42 MAP kinase and AKT in serum-starved or -stimulated gastric cancer cells. Total cell lysate was analyzed as described for figure c. The phosphorylation of p44/42 MAP kinase and AKT was detected by immunoblot using anti-phospho-specific antibodies. In each panel, the status of gene amplification and mutation (codon 12) of KRAS in each cell line is indicated. +, presence; -, absence.