Overexpression of uPAR increases P25-induced β1 integrin activation. Stable MG-63 cell lines transfected with either vector alone or vector containing the cDNA for human uPAR were grown to confluency in 24-well plates. The following day, cells were incubated for 2 hours in DMEM containing 50 μM P25 or S25. Supernatant was removed and cells were then incubated for 1 hour with 100 ng/ml of a monoclonal antibody (HUTS-4) which recognized either the active form of the β1 integrin (A) or another monoclonal antibody which recognized total β1 integrin (B). Cell layers were washed, fixed in paraformaldehyde, and incubated with a peroxidase conjugated secondary antibody. Bound antibody was detected by incubating cells with substrate, and color development was measured at A490 in a plate reader.