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Electroporation and drug selection of J1 ES cell clones were performed as described previously ( Figure 1Homologous recombination at the Vcam1 gene locus. (b) The wild-type murine Vcam1 gene, D4D targeting construct, and homologous recombination at the Vcam1 locus. (c) Southern blot analysis of Bam HI-digested genomic DNA from progeny of heterozygous mating.(a) Alternative RNA splicing produces VCAM-1 isoforms with one or two α4 integrin binding sites (solid circles). A solid line above the lanes indicates each litter. A common forward primer (5′-ATACATTGGGTATGGTGTGATATG) was used that bound to the 3′ end of exon 5 [phosphatidylinositol-linked (PI) domain].Chemokines then stimulate leukocytes and upregulate α4 and β2 integrin binding to VCAM-1 and ICAM-1, respectively, which themselves mediate later steps ().However, VCAM-1 is unique in that its expression is largely restricted to lesions and lesion-predisposed regions, whereas ICAM-1 expression extends into uninvolved aorta and lesion-protected regions ( mice express markedly reduced levels of VCAM-1 m RNA and protein consisting of 6 Ig domains (6 Ig VCAM-1), partially circumvent the embryonic lethality of complete VCAM-1 deficiency, and enable a direct comparison of VCAM-1 and ICAM-1 in atherosclerosis.RT (Super Script kit; Life Technologies Inc., Rockville, Maryland, USA) for alternatively spliced 7 and 6 Ig VCAM-1 used a primer complementary to GATTGAATTACTGAAGG in D5.For 3 Ig VCAM-1, the RT primer was complementary to CATCATTTGCATGGGGTCA in the PI domain.
The 382-, 217-, and 319-bp products for 7, 6, and 3 Ig VCAM-1, respectively, were resolved on 1.5% agarose gels.Cells were washed with PBS containing 2.5% FBS, and incubated with streptavidin-fluorochrome if required (4°C for 30 minutes), and erythrocytes were removed by hypotonic lysis.Flow cytometry was carried out immediately after the final wash using an Epics XL-MCL flow cytometer (Coulter Corp., Miami, Florida, USA).Plasma lipoprotein profiles were determined by gel filtration fast performance liquid chromatography ( A paired Student’s t test (Statview 4.5; Abacus Concepts Inc., San Francisco, California, USA) was used when data were obtained from paired littermates.Data from multiple groups were analyzed by ANOVA followed by Fisher’s protected least significant difference (PLSD) test to calculate pairwise comparisons.
) were depleted of macrophages using anti–Fc receptor magnetic beads.