Endothelial NF-κB blockade inhibited endothelial TF expression. (A-B) Endothelial NF-κB blockade diminished TNF-α–induced TF protein in ECs (A), but not LPS-induced TF protein in white blood cells (B). WT and TG ECs were treated with Dox (0.5 μg/mL) for 48 hours and then left untreated (WT-C and TG-C) or stimulated with TNF-α (100 ng/mL) for 6 hours (WT-T and TG-T). White blood cells were isolated from WT-Con (WT-C), WT-LPS (WT-L), TG-Con (TG-C), and TG-LPS (TG-L) mice at 6 hours after saline or LPS injection. TF band was not detected in proteins from control cells but induced in TNF-α–stimulated ECs (WT-T) or LPS-stimulated blood cells (WT-L). The TNF-α–induced TF band was prevented in TG ECs (TG-T). The LPS-induced TF band was not affected in TG white blood cells (TG-L). Blots are representative of 3 independent experiments. Actin indicates membrane for TF blotting was reblotted with actin antibody. (C-N) Representative immunofluorescence staining showing endothelial TF expression. Cryosections of kidney were prepared from mice at 6 hours after LPS injection and stained with anti-TF antibody (green) and anti-CD31 (an endothelial-specific marker) antibody (red). Positive staining for TF (E-F) was detected on sections from WT-LPS and TG-LPS mice. TF and CD31 double-positive staining (yellow) localizes TF-expressing ECs (M). TF-expressing ECs (yellow) were not detected in sections from WT-Con and TG-Con mice (K-L), increased in section from WT-LPS mice (M), and significantly reduced on section from TG-LPS mice (N). Bar represents 50 μm. Data are representative of 3 independent experiments. Slides were viewed with a confocal laser-scanning microscope system (FluoView 300-IX; Olympus) using a PLAN APO 60×/1.4 oil objective lens and VECTASHIELD Mounting Medium (Vector Laboratories). Images were processed and analyzed using Image J with colocalization plug-ins that statistically evaluate colocalization using Pearson correlation coefficient (r), which varies from −1 to 1, where 1 equals complete colocalization.