Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 Page 9 of 14 assay (Figure 8). In the presence of the p50-targeted antibody (but not for the p52-targeted reagent), there was a decrease in intensity of the more rapidly migrating EMSA signal, with a concomitant increase in intensity of the ‘super-shifted’ band (Figure 8). The ability of a-thrombin to trigger a PAR1-mediated activation of NF- B was thereby verified. Dynamism of mRNA expression of NF- B target genes induced by a-thrombin in PAR1-expressing cells We next evaluated the spectrum of NF- B target genes that might be activated by a-thrombin in MKN45/ PAR1. For this purpose, we selected 10 target genes of NF- B. A-thrombin treatment (15 nM, 3 hr) caused an increase in mRNA expression levels for all NF- B target genes tested, especially for TN-C, Bcl-2, cIAP1, and EGFR (Figure 9). The levels of TN-C, Bcl-2, cIAP1, and EGFR mRNA expression reached more than 100 times the corresponding levels of the control values in MKN45/PAR1. The increases in mRNA for these four NF- B-stimulated genes were mirrored by increases in the levels of the four corresponding proteins, as detected by western blot analysis (Figure 10). When PAR1 activa- tion was inhibited by SCH79797 or when NF- B activa- tion was inhibited by the CAPE (25 μg/ml), there was a substantial inhibition of the a-thrombin-triggered increase in the mRNA for these four NF- B target genes in MKN45/PAR1 (Figure 11). High molecular-weight factors resulting from PAR1 activation can activate EGFR in an autocrine manner We took notice of TN-C, because its mRNA expression presented the greatest magnitude of increase by PAR1 activation. ELISA results showed that the quantity of TN-C in the culture medium increased gradually up to 3 hr after the addition of a-thrombin and that the maxi- mum level was detected 12 hr after a-thrombin treat- ment in MKN45/PAR1 (Figure 12). A detectable Figure 10 Detection of the proteins, which were high expression mRNA under a-thrombin, by immunoblotting. MKN45/PAR1 were stimulated with 15 nM a-thrombin for 3, 6, and 12 hr and harvested for preparing whole cell lysates. The clear extracts containing 100 μg protein was developed on SDS-PAGE. The expression of TN-C, Bcl-2, cIAP1, EGFR and GAPDH protein, which was a house keeping gene product as a control, were detected by Western blotting using specific antibodies against respective proteins (The final dilutions of TN-C, Bcl-2, cIAP1 and EGFR antibodies were 1:500, GAPDH was 1:2000). The MKN45/PAR1 were harvested at 3, 6 and 12 hr after 15 nM a-thrombin stimulation and whole cells were lysed using LIPA buffer. Significant EGFR protein expression was detected in MKN45/mock and unstimulated MKN45/PAR1. The expression above basal level was clearly observed for TN-C, Bcl-2, cIAP1 and EGFR proteins 6 hr after addition of 15 nM a-thrombin.