Generation of wild-type and mutant (Ala380) PAI-2 expression plasmids.

(A) Schematic representation of the full-length wild-type (wt) and mutant (Ala380) PAI-2 cDNAs cloned into the multiple cloning site (MCS) of plasmid pCI-neo. The mutant PAI-2 expression plasmid contains an alanine substitution for arginine at the P1 residue at position 380 and is denoted as pCI–PAI-2_Ala380. E indicates exon. (B) PAI-2 expression and u-PA binding activity in COS cells transfected with either pCI-neo, pCI–PAI-2wt, or pCI–PAI-2_Ala380 was assessed by Western blot analysis using an anti–PAI-2 antibody. Extracts (100 μg) were incubated with u-PA (10 U) as indicated. As shown, cells transfected with the pCI–PAI-2wt plasmid produce a protein of 47 kd (lane 3) that forms SDS-stable complex with recombinant u-PA (lane 4). Cells expressing mutant PAI-2, although producing a protein of a similar molecular weight (lane 5), cannot form SDS-stable complexes with u-PA (lane 6). Lanes 1 and 2: extracts prepared from COS cells transfected with the control pCI-neo plasmid.