Induction of HHcy enhances expression of TF. (a) HUVECs were exposed to the indicated concentration of BSA, L-HC, or L-cysteine for 8 hours. Cells were harvested and prepared for immunoblotting using goat anti-rat TF IgG (0.5 μg/ml). ^AP < 0.05 vs. lanes 1 and 3. (b–h) ApoE-null mice were sacrificed after 8 weeks of the indicated diet, aortae were removed, and lysates were prepared. Protein (10 μg) was subjected to SDS-PAGE and transfer to nitrocellulose. Immunoblotting was performed with goat anti-rat TF IgG (1 μg/ml). Molecular weight markers are indicated. Densitometric analysis was performed; pixel units from aortic tissue derived from mice receiving diet A or BSA-treated HUVECs were arbitrarily assigned a relative value of 1. ^BP < 0.01 vs. diets A and C. In b, immunoblotting on lysates from n = 10 mice per diet was performed; representative experiments are shown. In c–h, apoE-null mice were fed diet A (c, f), diet B (d, g), or diet C (e, h), for 8 weeks. Upon sacrifice, serial sections at the aortic sinus were prepared and stained with anti–Mac-3 IgG (c–e) (10 μg/ml) or goat anti-rat TF IgG (f–h) (10 μg/ml). In c–h, immunohistochemistry was performed on n = 5 mice/diet; representative experiments are shown. Scale bar, 50 μm.