Activation of NF-κB assessed by EMSA analysis in MKN45/mock and MKN45/PAR1 by α-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 α-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-κB cis-element ds-oligo probe on the gel. An arrow on left side of the middle figure indicates the NF-κB/DNA binding complex. In the super-shift assay, antibodies (2 μg) raised against NF-κBp50 and NF-κBp52 were mixed with nuclear extract obtained from PAR1-transfectanted cells treated with 15 nMα-thrombin for 0.5 hr. A super-shifted band was clearly detected with NF-κBp50 antibody as marked by an arrowhead, but not detected with NF-κBp52 antibody.