Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 8 of 14 Figure 8 Activation of NF- B assessed by EMSA analysis in MKN45/mock and MKN45/PAR1 by a-thrombin stimulation. The gel mobility shift assay was performed on the whole nuclear extract from MKN45/mock and MKN45/PAR1 (in a 12 cm diameter dish) that were incubated in the presence of 15 nM a-thrombin for 0.5, 1, 2, 6, and 24 hr. MKN45/mock samples showed no retardation bands but only intense broad signal due to biotinylated NF- B cis-element ds-oligo probe on the gel. An arrow on left side of the middle figure indicates the NF- B/DNA binding complex. In the super-shift assay, antibodies (2 μg) raised against NF- Bp50 and NF- Bp52 were mixed with nuclear extract obtained from PAR1- transfectanted cells treated with 15 nMa-thrombin for 0.5 hr. A super-shifted band was clearly detected with NF- Bp50 antibody as marked by an arrowhead, but not detected with NF- Bp52 antibody. Figure 9 The mRNA expression of NF- B target genes by quantitative RT-PCR. We selected 10 target genes of NF- B that are concerned with cell growth and invasion. Most of the mRNA expression were increased after 15 nM a-thrombin stimulation. The mRNA expression levels of TN-C, Bcl-2, cIAP1 and EGFR reached more than 100 times the relative levels of the control values. Especially, TN-C mRNA expression level was increased. The kinetics of mRNA expression following a-thrombin stimulation in MKN45/PAR1 are different depending upon type of genes tested. The bar height indicates the calculated fold activation values for each time after stimulation with 15 nM a-thrombin. It was based on the condition of no a-thrombin stimulation. (Date were expressed as mean values ± SD from triplicate experiments.)