Induction of HHcy enhances vascular expression of VCAM-1 and RAGE. (a–d) VCAM-1. ApoE-null mice were fed the indicated diet for 8 weeks. Aortae were removed, and lysates were prepared. Lysate protein (10 μg) was subjected to immunoblotting with goat anti–VCAM-1 IgG (1 μg/ml). In a, ^AP < 0.05 vs. lanes 1 and 3. (b–d) Immunohistochemistry was performed using anti–VCAM-1 IgG (8 μg/ml). (e–i) RAGE. In e, HUVECs were exposed to BSA, L-HC, or L-cysteine (100 μM) for 8 hours. Lysates were prepared and subjected to immunoblotting with anti-RAGE IgG (2 μg/ml). ^AP < 0.05 vs. lanes 1 and 3. In f, immunoblotting of aortic lysates was performed using anti-RAGE IgG (2 μg/ml). ^AP < 0.05 vs. lanes 1 and 3. (g–i) Immunohistochemistry was performed using anti-RAGE IgG (20 μg/ml). In a, e, and f, molecular weight markers are indicated on the right side of each blot. Densitometric analysis was performed; pixel units from aortic tissue derived from mice receiving diet A or HUVECs exposed to BSA were arbitrarily assigned a relative value of 1. In a and f, immunoblotting was performed on n = 7 mice/diet; representative experiments are shown. In b–d and g–i, immunohistochemistry was performed on n = 5 mice/diet; representative experiments are shown. Scale bar, 50 μm.