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CHOB2-α5 and CHOK1 cells (provided by A. Rick Horwitz, University of Virginia) were maintained in DMEM containing 10% fetal bovine serum (FBS) (Invitrogen), l-glutamine, nonessential amino acids, and antibiotics. The α5β1 integrin engages the central cell binding domain (CCBD) https://trexminer.info/ of FN and forms focal adhesions, which function both as anchors and as signaling centers. In particular, the fibronectin (FN) binding integrins α4β1 and α5β1 are well characterized for their roles in cell motility.
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CHOK1 cells that express an α4/GFP chimera as well as endogenous α5β1 integrin (CHO-α4/GFP), also bound to the ADAM12 disintegrin domain, and this interaction was disrupted by the anti-α4 antibody (Figure 1C). Binding of CHOB2-α4 cells was inhibited by an antibody against the α4, but not one against the α5, integrin subunit (Figure 1A). In contrast, CHOB2-α5 cells, which express the human α5 integrin and bind to fibronectin (FN) (Figure 1B), did not adhere to the ADAM12 disintegrin domain (Figure 1A). 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).