Figure 4.

FGF-2–treated fibroblasts expressed more full-length uPAR than TGFβ-treated fibroblasts. By immunoblot detection and immunofluorescence, we found that cells grown for 72 h with FGF retained significantly more full-length uPAR compared with TGFβ-treated cells. Keratocytes (quiescent fibroblasts) did not express uPAR (n = 5; results from 5 different cornea donors). (A) Cell lysates were detected for the presence of total uPAR by Western blot (n = 10; results from 5 different cornea donors). Freshly isolated keratocytes (lane 1), fibroblasts plated in the presence of FGF (lanes 2 and 4), or TGFβ (lanes 3 and 5). (B) Cells were seeded in SSFM-FGF, SSFM-TGFβ, or SSFM alone, and they were grown for 72 h. To remove any uPA-bound uPAR that could block uPAR antibody binding, cells were stripped of cell surface uPA with 0.05 M glycine and 0.1 M NaCl, pH 3.0, before fixation, followed by washing with PBS (Stoppelli et al., 1986; Baker et al., 1992; Bernstein et al., 2004). To detect full-length uPAR, an antibody to the D1 domain of uPAR was used (see Materials and Methods). Total uPAR was detected with an antibody to the D2 domain, which is present in both full-length and cleaved uPAR (see Materials and Methods). Bar, 20 μm. (C) To quantify the relative amounts of full-length uPAR to total uPAR, colocalized images were captured with identical parameters and analyzed for their intensity using MetaMorph image analysis software. For each condition, ∼20 fields from two different cornea donors were imaged and analyzed.