Expression of wild-type and mutant PAI-2 in THP-1 cell clones.

Cytoplasmic extracts (100 μg) prepared from nontreated THP-1 cells (clones a-d) expressing wild-type PAI-2 (wt PAI-2, lanes 2-5) and from cells expressing mutant PAI-2 (mut PAI-2, clones e-h) (lanes 6-9) were subjected to SDS-PAGE analysis. Relative levels of PAI-2 protein were assessed by Western blot analysis using a monoclonal anti–PAI-2 antibody. As shown in the upper panel, PAI-2 protein is present in all clones expressing either wild-type or mutant PAI-2. Lane 1: extracts prepared from pCI-neo–transfected cells. The broken arrow to the right of the figure indicates nonspecific (NS) binding. To determine whether PMA could further increase PAI-2 protein, all clones of THP-1 cells expressing active or mutant PAI-2 were treated with 25 nM PMA for 24 hours. Cytoplasmic extracts (20 μg) were prepared and subjected to SDS-PAGE and Western blot analysis as described above. As shown in the lower panel, PMA treatment caused at least a 5-fold increase in PAI-2 protein in all clones of cells expressing either wild-type or mutant PAI-2.