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An experiment with cells transfected to express intracellular GFP showed that the biotinylation protocol did not label intracellular proteins (our unpublished data). Migration tracks of individual cells were analyzed using Openlab 2.2.5 software. Briefly, 35-mm glass bottom culture dishes (MatTek) were precoated with 2 μg/ml CS-1 overnight at 4°C, and 5 × 105 mock or ADAM12-transfected cells were plated on a dish for 2 h.

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Cells expressing α4β1 but not α5β1 integrin bind to recombinant ADAM12 disintegrin domain. A previous study demonstrated that GFP-tagged α4 integrin behaves like untagged α4 integrin (Pinco et al., 2002). Transwell migration assays were performed as described previously (Liu et al., 1999), with the following modifications. After incubation for 2 h in a CO2 incubator at 37°C, confluent cell monolayer was scraped with a yellow Pipetman tip to generate scratch wounds.

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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). For each experiment, the average migration rate of ADAM12-transfected cells was normalized to that of mock-transfected cells. For transwell migration assays, the distal membranes of transwell inserts were coated with 10 μg/ml FN, and number of cells that migrated through the pores were determined after an incubation of 15 h (for CHOB2-α4 and CHOB2-α5 cells) or 4 h (for CHO-α4/GFP and CHOK1 cells). We next tested the effect of expressing full-length ADAM12 on directed cell migration on FN, using both transwell and scratch wound assays. (C and D) CHO-α4/GFP (black) and CHOK1 cells (white) were pretreated with either no http://www.ahmetozhan.com.tr/ antibody or 5 μg/ml anti-α4 antibody (HP2/1) for 30 min before adding to wells coated with 5 μg/ml ADAM12 disintegrin domain (C) or human plasma FN (D).

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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. Twenty-four hours after the transfection, cells were labeled with either a control antibody, or an antibody against the integrin α4 subunit (HP2/1), followed by phycoerythrin-conjugated goat anti-mouse IgG. 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.