Total RNA was isolated from cell cultures and mouse aortic arch (third rib to aortic root). Consecutive mouse aortic root sections were incubated with goat anti-LDB2, rat monoclonal anti-mouse CD68, rabbit polyclonal anti-mouse SM22 alpha, or rabbit polyclonal anti-human VWF at 4°C overnight and counterstained with hematoxylin. RT–PCR was performed on total RNA isolated from human pulmonary artery SMCs, THP-1 monocytes, THP-1 macrophages generated with phorbol 12-myristate 13-acetate, THP-1 foam cells cultured from THP-1 macrophages incubated with acetylated low density lipoproteins, primary macrophages differentiated from primary monocytes isolated from human blood with AB serum, cultured EAHY926 cells, EAHY926 cells induced with 20-ng/ml human recombinant TNF-α, and HUVECs isolated with collagenase. (A) Mouse LDB2 and VWF protein expression in serial sections of aortic roots from Ldlr−/−Apob100/100 mice at 10 weeks (arterial wall without visual atherosclerosis, “non-atherosclerotic”), 20 weeks (early lesions, fatty streaks), and 50 weeks (late lesion, plaques). Ovals indicate areas of overlapping LDB2 and VWF staining in relation to negative controls. (B) LDB2 mRNA levels in EAHY926 cells, induced EAHY926 cells, and HUVECs (n = 4 per cell type; scales on Y-axes are comparable because the RT-PCR was performed in one single run). (C) Mouse LDB2, CD68, and SM22 alpha protein expression in serial sections of aortic roots from Ldlr−/−Apob100/100 mice at 20 and 50 weeks. (D) LDB2 mRNA levels in primary human SMCs, THP-1 monocytes, THP-1 monocytes differentiated into THP-1 macrophages, THP-1 foam cells, and primary human monocytes differentiated into macrophages (n = 4 per experiment). Ovals indicate areas of overlap between LDB2 and CD68 but no or very subtle SM22 staining in relation to negative controls. (E) mRNA levels measured by real-time PCR from late (40 weeks, plaques, n = 5) and early (20 weeks, fatty streaks, n = 5; lesions from the aortic arch in Ldlr−/−Apob100/100 mice.