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. 2000 Jun 19;191(12):2053–2064. doi: 10.1084/jem.191.12.2053

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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(continued on facing page). Expression of CD44v7 and generation of CD44v6/v7^−/− mice. (A) Immunohistochemical analysis of CD44v7 isoforms in inflamed mucosa showing expression of slightly inflamed mucosa (top left) as well as in large transmural infiltrates (bottom right). IM7.8.1 (anti–pan-CD44) reveals broad staining (bottom left). Original magnifications: ×200; negative control, ×100. (B) cDNA was prepared from LPMCs of noninflamed and inflamed large intestines of TNBS colitis, and semiquantitative RT-PCR of CD44 variant isoforms was performed using primers I and II located within the standard region, flanking the variant part. Amounts of cDNA were equilibrated to HPRT- and CD44-specific reactions blotted and hybridized with exon-specific probes. Reactions for v3, v7, v10, and the standard region (CD44s) are shown in the bottom panel. Presence and size of a signal allowed for the composition of the variant isoforms expressed, which are indicated in the scheme above. LPMCs from inflamed colons express a variety of CD44v isoforms, mostly including v7, whereas LPMCs from noninflamed tissue express exons v3 and v10 as a single exon, as well as v10 in combination with v8 and v9. The gray bars in the upper region of the scheme represent the CD44 standard region, which is expressed with similar intensity in all preparations. (C) Genomic targeting of CD44 exons v6 and v7. Analysis of ES clones by Southern blotting using a 5′ external probe (StuI–EcoRI; probe A). Southern blot analyses using probe B (EcoRI–EcoRV) indicated loss of 1.6 kb in the region of exon v7, which only occurred in one of the two positive ES clones. The restriction sites are: St, StuI; R, EcoRI; BE, BstEII; S, SacI; BX, BstXI; Bst, Bst1107I; V, EcoRV; and B, BamHI. (D) LN cells were prepared and stimulated overnight with PHA or cocultured with CD40L-transfected J558 cells in transwell plates (Costar). Surface staining was performed using pan-CD44–specific mAb (clone IM7.8.1)–FITC, and biotinylated CD44v6 (LN6.1 or BMS145; Bender MedSystems) specific and v7 (LN7.1) specific antibodies. Avidin-PE was used for detection of the CD44v expression. Percentage of double labeled cells is indicated in the upper right quadrant.

(continued on facing page). Expression of CD44v7 and generation of CD44v6/v7^−/− mice. (A) Immunohistochemical analysis of CD44v7 isoforms in inflamed mucosa showing expression of slightly inflamed mucosa (top left) as well as in large transmural infiltrates (bottom right). IM7.8.1 (anti–pan-CD44) reveals broad staining (bottom left). Original magnifications: ×200; negative control, ×100. (B) cDNA was prepared from LPMCs of noninflamed and inflamed large intestines of TNBS colitis, and semiquantitative RT-PCR of CD44 variant isoforms was performed using primers I and II located within the standard region, flanking the variant part. Amounts of cDNA were equilibrated to HPRT- and CD44-specific reactions blotted and hybridized with exon-specific probes. Reactions for v3, v7, v10, and the standard region (CD44s) are shown in the bottom panel. Presence and size of a signal allowed for the composition of the variant isoforms expressed, which are indicated in the scheme above. LPMCs from inflamed colons express a variety of CD44v isoforms, mostly including v7, whereas LPMCs from noninflamed tissue express exons v3 and v10 as a single exon, as well as v10 in combination with v8 and v9. The gray bars in the upper region of the scheme represent the CD44 standard region, which is expressed with similar intensity in all preparations. (C) Genomic targeting of CD44 exons v6 and v7. Analysis of ES clones by Southern blotting using a 5′ external probe (StuI–EcoRI; probe A). Southern blot analyses using probe B (EcoRI–EcoRV) indicated loss of 1.6 kb in the region of exon v7, which only occurred in one of the two positive ES clones. The restriction sites are: St, StuI; R, EcoRI; BE, BstEII; S, SacI; BX, BstXI; Bst, Bst1107I; V, EcoRV; and B, BamHI. (D) LN cells were prepared and stimulated overnight with PHA or cocultured with CD40L-transfected J558 cells in transwell plates (Costar). Surface staining was performed using pan-CD44–specific mAb (clone IM7.8.1)–FITC, and biotinylated CD44v6 (LN6.1 or BMS145; Bender MedSystems) specific and v7 (LN7.1) specific antibodies. Avidin-PE was used for detection of the CD44v expression. Percentage of double labeled cells is indicated in the upper right quadrant.