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Twenty-four hours after transfection, cells were labeled with the membrane-impermeable EZ-Link NHS-LC-biotin (Pierce Chemical), and avidin precipitation and immunoblotting were conducted as described in Materials and Methods. CHO-α4/GFP cells were transfected with 4 μg of DNA (per 6-cm dish) encoding the indicated 6Xmyc-tagged constructs. Expression of wild-type and mutant full-length ADAMs on the surface of CHO-α4/GFP cells. Average wound closure for cells transfected with different ADAM12 constructs was expressed as a percentage of that seen for https://www.daihatsumakassar.co/ mock-transfected cells.
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Mock-transfected cells at the wound edge protruded broad lamellipodia and displayed polarity toward the wound (Figure 4B, left, and Movie 1A). Microscopy of mock- or ADAM12-transfected CHO-α4/GFP cells in scratch wound migration assays. (A) CHO-α4/GFP cells, which express both the α4β1 and α5β1 integrins, were either mock transfected or transfected to express full-length ADAM12. Full-length ADAM12 inhibits the migration of CHO-α4/GFP cells on the α4β1 binding CS-1 region but not on the α5β1 binding CCBD of fibronectin. In contrast, expression of ADAM12 had no significant effect on the migration of the same cells (CHO-α4/GFP) on the α5β1 substrate (Figure 3C).
A similar observation with the D488A mutant was obtained using CHOB2-α4 cells in adhesion flow assays. Adhesion assays were then conducted using Molt 3 human lymphoma cells as described in the legend to Figure 1, except that 1 mM Mn2+ was added to cell suspensions before the assays. Of these charge to alanine mutations, the mutant D488A was strongly impaired in its ability to support α4β1-mediated cell adhesion (Figure 5A). Recombinant mutant disintegrin domains were then tested for their ability to support integrin α4β1-dependent cell adhesion. Percentage of positive cells and mean fluorescence units (MFU) for both phycoerythrin and GFP were determined by flow cytometry.
