Figure 7.

Overexpression of PAG3, but not its GAP-inactive mutant, decreases the cell migratory activity without significant effect on the cell adhesive activity. COS-7 cells or U937 cells were transiently transfected with plasmids encoding EGFP-tagged PAG3 (WT) or the CA mutant (CA) or subjected to the transfection procedure without plasmid DNA (Mock). U937 cells were treated with TPA for 3 d. Cells were collected by trypsinization and then subjected to haptotaxis migration assays (A) or adhesion assays (B) on collagen (Col), fibronectin (FN), or vitronectin (VN) in the absence of serum as described in MATERIALS AND METHODS. BSA coating was used as a negative control. Transfected cells adhered 30 min after replating were enumerated by counting cells positive for the EGFP fluorescence, and cells migrating during 3 h were enumerated by counting cells on the underside of the membrane that were positive for the EGFP fluorescence. Percentages of cell adhesion and migration were calculated as described in MATERIALS AND METHODS. Each bar represents the mean ± SEM of triplicate experiments. The inset in A is the anti-GFP antibody immunoblot showing the expression levels of EGFP-tagged PAG3 (WT) or the CA mutant (CA) in COS-7 cells used in these experiments. Expression of EGFP-tagged PAG3 protein was >10–20 times higher than that of endogenous PAG3 (our unpublished data).