Figure 14

Detection of phosholyrated EGFR by TN-C. The conditioned medium of PAR1/MKN45 culture pre-stimulation by 15 nM α-thrombin for 3 and 12 hr was collected and partially purified by repeated concentration using ultra-filtration spin columns able to cutoff macromolecules below 200 kDa. MKN45/PAR1 were incubated either with the concentrated conditioned medium for 6 hr, which were pre-stimulated for 3 hr or 12 hr by 15 nM α-thrombin. These whole lysates of MKN45/PAR1 treated with the conditioned medium pre-stimulated for 12 hr by 15 nM α-thrombin was detected remarkably enhanced tyrosine phospholyration of the EGFR, but these whole lysates of MKN45/PAR1 with the conditioned medium pre-stimulated for 3 hr by 15 nM α-thrombin was not detected the tyrosine phospholyration of the EGFR (left).

The whole lysates of MKN45/PAR1 treated with the concentrated conditioned medium with TN-C-neutralizing antibody (25 μg/ml), which was pre-stimulated for 12 hr by 15 nM α-thrombin, show no-increased in phosphor-EGFR protein quantity (right).