Functional assessment of PAI-2 expressed in THP-1 cells.

(A) To determine whether the wild-type PAI-2 expressed in THP-1 cells retained u-PA binding activity, cytoplasmic extracts (30 μg) prepared from THP-1 cells expressing wild-type PAI-2 (clone c) were incubated with increasing concentrations of u-PA (0-5 U) as indicated at the top of the figure. PAI-2 protein was detected by Western blot analysis using a monoclonal anti–PAI-2 antibody. As shown, addition of u-PA resulted in complex formation that was evident when using 1 U u-PA (lane 5). Complete displacement of PAI-2 was obtained with 5 U u-PA (lane 7). The same procedure was performed using cytoplasmic extracts (30 μg) prepared from THP-1 cells expressing mutant PAI-2 (clone g). No complex formation is with u-PA even at the highest concentration of u-PA added (lower panel). (B) Cytoplasmic extracts (30 μg) prepared from all clones of THP-1 cells expressing wild-type PAI-2 (clones a-d) and all clones of cells expressing mutant PAI-2 (clones e-h) were incubated with 2 U u-PA. Samples were then applied to SDS-PAGE and PAI-2 protein detected by Western blot analysis as described above. As shown, all clones of cells expressing wild-type PAI-2 contained active inhibitor, as evidenced by the formation of SDS-stable complex formation (lanes 2-5). In contrast, all clones expressing mutant PAI-2 failed to produce a complex with u-PA (lanes 6-9). Lane 1: extracts prepared from cells transfected with pCI-neo.