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As seen in Figure 1A, CHOB2 cells that express the human α4 integrin subunit (CHOB2-α4), bound to a recombinant ADAM12 disintegrin domain. We also tested whether cells expressing α5β1 integrin adhere because we are interested in the selectivity of ADAM-integrin interactions, and because very few ADAM disintegrin domains have been reported to interact with α5β1 integrin (White et al., 2005). ADAM12 is phylogenetically related to Xenopus ADAM13, which has a documented role in cranial neural crest (CNC) cell migration (Alfandari et al., 2001). To date, only a few studies have addressed the possible role of ADAM-integrin interactions using full-length ADAM proteins in cell biological settings. Integrins often work in cooperation with syndecans, transmembrane proteoglycans that foster cell adhesion, stress fiber formation, cell migration, and invasion (Woods and Couchman, 2000; Beauvais and Rapraeger, 2004).
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Sequences encoding the disintegrin domains of mouse ADAM19 (residues G417-G504) or mouse ADAM 33 (residues P418-G505) in frame with a terminal 6Xhistidine tag were generated by PCR. ADAM12 disintegrin loop mutants (R481A, D488A, and E491A), were generated using a site-directed mutagenesis kit (Stratagene), using the 6XHis-tagged ADAM12 disintegrin domain as the template. To produce protein with higher purity, a 6Xhistidine (His) tag was added to the 3′ end of the disintegrin domain coding sequence, using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mouse ADAM12 disintegrin domain in the vector pGEX-2T was provided by Anna Zolkiewski (Kansas State University, Manhattan, KS). Mouse anti-α4 integrin functional blocking monoclonal antibody (mAb) HP2/1 and mouse anti-α5 integrin functional blocking antibody CBL497 were obtained from Chemicon International (Temecula, CA). Many recombinant ADAM disintegrin domains have been shown to interact with a variety of β1 integrins, including α4β1 (Tomczuk et al., 2003; White, 2003; Bridges et al., 2004; White et al., 2005).
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Pinco Casino delivers a polished lobby packed with hit titles, engaging features, and generous promotions tailored for slot lovers. This work was supported by National Institutes of Health Grant GM (to J.M.W.). Further studies will investigate the mechanisms and consequences of this newly appreciated ADAM function. In contrast, as noted above, the disintegrin domain seems to be important for this activity. Further genetic experiments suggested that UNC-71 acts together with integrins for proper axon guidance (Huang et al., 2003). We studied ADAMs 12, 19, and 33 because they are phylogenetically related to an ADAM (Xenopus ADAM13) that functions in Xenopus cranial neural crest migration (Alfandari et al., 2001), and because they are expressed in mouse cranial neural crest (Tomczuk, 2004).