Figure 3

Effect of uPAR expression levels on P25-induced fibronectin matrix assembly. A – Lysates were prepared from vector (V) or uPAR transfected (uPAR) stable lines of MG-63 cells. uPAR levels were analyzed by Western blotting with anti-uPAR antibody. The membrane was stripped and reprobed with anti β-actin antibody as a loading control. B – Vector and uPAR transfected cell lines were cultured overnight and then stimulated with 50 μM S25 or P25 for 6 h in the presence of ¹²⁵I-fibronectin. Cell layers were extracted in 1% DOC, and ¹²⁵I-fibronectin that was incorporated into the detergent-insoluble matrix fibronectin was recovered by centrifugation and measured by gamma scintillation. C – uPAR-/- MEF were transiently transfected with the cDNA for human uPAR. 24 hours after transfection, MEFs were washed and monolayers were stimulated with 50 μM S25 or P25 for 6 hours in the presence of ¹²⁵I-fibronectin. Cell layers were extracted in 1% DOC, and ¹²⁵I-fibronectin that was incorporated into detergent-insoluble matrix was recovered by centrifugation and measured by gamma scintillation. Control cells received no added peptide.