FIG. 4.

PAI-1 inhibits LNCaP and PC3 processing of full-length PDGF D. (A) PC3-PDGF D cells were plated in a 48-well plate and grown to 100% confluence. Cells were then washed with PBS, coinfected with recombinant PDGF D virus and vTF7-3 for 30 min (see Materials and Methods), washed with PBS, and then cultured in serum-free medium with the indicated protease inhibitors or vehicle controls (EtOH or dimethyl sulfoxide [DMSO]) for 48 h. CM was then collected and resolved on a reducing SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with our PDGF D antibody. FL-M, full-length PDGF D protein monomer; GD-M, PDGF D growth domain (GD) monomer. (B) LNCaP-PDGF D cells were treated as described above, except infected with vTF7-3 virus control only. CM was collected and analyzed as described above. (C) PC3-PDGF D cells were plated in a six-well plate to 100% confluence, washed with PBS, and then incubated in serum-free medium with 10 μg/ml of aprotinin or 200 nM of PAI-1 for 48 h. Media were collected, and PDGF D was concentrated using Ni-NTA agarose beads. Samples were resolved on an SDS-PAGE gel under reducing conditions and transferred to a nitrocellulose membrane, and PDGF D was detected using anti-PDGF D-GD antibody.