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. 2005 Jul;25(14):6279–6288. doi: 10.1128/MCB.25.14.6279-6288.2005

FIG. 5.

FIG. 5.

PDGF D is a substrate of uPA. (A) rPDGF D was incubated overnight with 10 nM of indicated protease, resolved on an SDS-PAGE gel under reducing conditions, and analyzed by immunoblotting using anti-PDGF D-growth domain (GD) antibody. FL-M, full-length PDGF D protein monomer; GD-M, PDGF D growth domain monomer. (B) rPDGF D was incubated with increasing nanomolar amounts of recombinant uPA and incubated at 37°C overnight. Samples were analyzed by immunoblotting using the anti-PDGF D-GD antibody. (C) Five milliliters of CM was collected from CV-1 cells infected with vaccinia virus-expressed rPDGF D and vTF7-3 or vTF7-3 only and was incubated with no uPA (lane 3 and lane 6) or 10 nM uPA (lane 4 and lane 7) overnight and then used to stimulate serum-starved NIH 3T3 cells for 10 min. In addition to the vTF7-3 control, additional negative controls included NIH 3T3 cells stimulated with serum-free (SF) medium (lane 1) or SF medium incubated overnight with 10 nM uPA (lane 5). β-PDGFR was immunoprecipitated by incubating 500 μg of lysate with anti-β-PDGFR antibody. Samples were resolved on an SDS-PAGE gel under reducing conditions, transferred to a nitrocellulose membrane, and then probed with an antiphosphotyrosine antibody. As a control of the immunoprecipitation efficiency, the membrane was stripped and reprobed with an anti-β-PDGFR antibody.