NF-κB activation resulted in reduced cellular levels of EPCR and TM in cultured ECs. WT and TG ECs were incubated with Dox (0.5 μg/mL) for 48 hours to induce I-κBαmt expression in TG ECs, and left untreated (WT-C and TG-C) or stimulated with 100 ng/mL TNF-α (WT-T and TG-T) for 1 (for measuring NF-κB activity) or 14 hours (for detecting EPCR and TM proteins). Bolts are representative of 3 experiments. (A) Representative Western blot photograph showing NF-κB activation measured by p65 nuclear translocation. TNF-α markedly activated NF-κB in WT ECs (WT-T), which was significantly inhibited in TG ECs (TG-T). (B) Representative Western blot photograph showing that TNF-α stimulation decreased cellular level of EPCR protein in WT ECs (WT-T), but not in TG ECs (TG-T). (C) Representative Western blot photograph showing that TNF-α stimulation decreased cellular level of TM protein in WT ECs (WT-T), but not in TG ECs (TG-T). Actin indicates membrane for p65, EPCR, or TM blotting was reblotted with actin antibody.