Induction of TF by OxPAPC is independent of the NF-κB pathway.

HUVECs were treated with OxPAPC (125 μg/mL) or TNF-α (100 U/mL) for indicated times and then processed for Western blotting with antibodies to phospho-IκBα (A) or total IκBα (B). (C) HUVECs were stimulated with OxPAPC (125 μg/mL) or TNF-α (25 U/mL) for 1 hour. Cells were scraped and activated p65 was detected in cell lysates using an ELISA-based kit Trans-am NF-κB p65. The data are presented as means ± SD of triplicate measurements. Similar results were obtained in an independent experiment. (D) HUVECs were cotransfected with pNF-κB-Luc and pCMV-β and after 48 hours stimulated with OxPAPC (120 μg/mL) or LPS (250 ng/mL). After 6 hours the cells were scraped and analyzed for luciferase and β-galactosidase activities. The results are expressed as the ratio between luminescence units in luciferase assay and optical density in β-galactosidase assay. The data represent mean values ± SD. Similar results were obtained in an additional experiment. (E) HUVECs were infected either with control (AdGFP) or AdIκBα, and 24 hours after infection stimulated with OxPAPC (125 μg/mL) or TNF-α (100 U/mL) for 6 hours. TF activity was determined by clotting assay from duplicate wells. Similar results were obtained in an independent experiment.