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. 2006 Jun 12;203(6):1603–1614. doi: 10.1084/jem.20052543

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Copyright © 2006, The Rockefeller University Press

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Figure 2. — Cell and tissue expression of nepmucin. (A) Nepmucin mRNA expression analyzed by RT-PCR. Left: HEV cDNA libraries (PNAd⁺ HEVs and MAdCAM-1⁺ HEVs), endothelial cell lines (KOP2.16, SVEC4-10, bEND3, and F2), monocyte/macrophage cell lines (WEHI3B, MH-S, and P388D1), and a mast cell line (P815) were analyzed. Right: Freshly isolated MAdCAM-1⁺ HEV cells, T cells, B cells, and DCs were analyzed. The PCR primers were designed to detect all the nepmucin isoforms as a single band. (B) Expression of nepmucin splicing isoforms in freshly isolated MAdCAM-1⁺ HEVs. A primer pair designed to detect nepmucin isoforms with different product sizes was used. (C) Tissue distribution of nepmucin. The expression of nepmucin was analyzed by Western blotting using the anti-nepmucin mAb ZAQ2, which recognized all four nepmucin variants (arrows). Control rat IgG2a did not give any specific signals (unpublished data). (D) Enzymatic O-deglycosylation of nepmucin. Lysates from mouse heart were subjected to immunoprecipitation with ZAQ2 and mock-treated (lane 1) or treated with O-glycanase (lane 2). The proteins were analyzed with the anti-nepmucin mAb, ZAQ3. The arrows and arrowheads indicate the four kinds of antigenic components.