Figure 4.
Figure 4. FGF-R1/PDGF-Rα interaction examined in SPA and coimmunoprecipitation. (A) Solid-phase assay (SPA) was performed to analyze PDGF-Rα binding to immobilized FGF-R1. Up to 100 nM biotinylated-PDGF-Rα recombinant protein, in the presence of 10 M excess of unlabeled PDGF-Rα, was incubated onto immobilized FGF-R1 (10 μg/mL), and a colorimetric assay was carried out. Specific binding was calculated by subtracting nonspecific from total binding. Bmax and Kd values were computed by GraphPad Prism 4 software, applying a nonlinear regression fit. Scatchard analysis is also reported (inset). (B) Interaction of soluble PDGF-Rα (800 nM each) to plastic (□) or to FGF-R1–immobilized factor (28 μg/mL) (▪) is reported as OD. Left: PDGF-Rα interaction in the absence of ligands. Right: PDGF-Rα interaction in the presence of ligands. Results are reported as mean ± SD of 3 experiments. *P < .05 (▪ vs □). #P < .05 (▪ vs ▪). (C) Lysates of PAE cells transfected with human PDGF-Rα were immunoprecipitated with anti–PDGF-Rα antibody and analyzed by immunoblotting, probed with anti–FGF-R1 antibody (bottom bands), and reprobed with anti–PDGF-Rα antibody (top bands). FGF-R1/PDGF-Rα coimmunoprecipitation was found in the absence (lane 2) and in the presence (lane 3) of FGF-2/PDGF-BB. Lane 4 reports PAE cells transfected with pcDNA3 empty vector, as control. They expressed very low levels of PDGF-Rα; thus, no signal was evident in Western blot for PDGF-Rα or for FGF-R1. Lane 1 represents the positive control for FGF-R1; it shows Western blot of cell lysates immunoprecipitated and immunoblotted with anti–FGF-R1 antibody and allows identification of FGF-R1 as the band observed in lanes 2 and 3. PDGF R-α was not detected in lane 1 as opposed to lanes 2 and 3, likely for a different steric impediment of the antibody couple used in lane 1 compared with the antibody couple used in lanes 2 and 3. (D) Lysates of untransfected HUVECs were immunoprecipitated with anti–PDGF-Rα antibodies and were analyzed by immunoblotting, probed with anti–FGF-R1 antibody (bottom bands), and reprobed with anti–PDGF-Rα antibodies (middle bands). FGF-R1/PDGF-Rα complex was found in untreated (lane 1) and FGF-2/PDGF-BB–treated (lane 2) cells. Immunoblotting with antiphosphotyrosine antibody (top bands) shows the activation of PDGF-Rα in lane 2. Lane 3 shows expression levels of FGF-R1 and PDGF-Rα by Western blot analysis of a cellular HUVEC lysate.

FGF-R1/PDGF-Rα interaction examined in SPA and coimmunoprecipitation. (A) Solid-phase assay (SPA) was performed to analyze PDGF-Rα binding to immobilized FGF-R1. Up to 100 nM biotinylated-PDGF-Rα recombinant protein, in the presence of 10 M excess of unlabeled PDGF-Rα, was incubated onto immobilized FGF-R1 (10 μg/mL), and a colorimetric assay was carried out. Specific binding was calculated by subtracting nonspecific from total binding. Bmax and Kd values were computed by GraphPad Prism 4 software, applying a nonlinear regression fit. Scatchard analysis is also reported (inset). (B) Interaction of soluble PDGF-Rα (800 nM each) to plastic (□) or to FGF-R1–immobilized factor (28 μg/mL) (▪) is reported as OD. Left: PDGF-Rα interaction in the absence of ligands. Right: PDGF-Rα interaction in the presence of ligands. Results are reported as mean ± SD of 3 experiments. *P < .05 (▪ vs □). #P < .05 (▪ vs ▪). (C) Lysates of PAE cells transfected with human PDGF-Rα were immunoprecipitated with anti–PDGF-Rα antibody and analyzed by immunoblotting, probed with anti–FGF-R1 antibody (bottom bands), and reprobed with anti–PDGF-Rα antibody (top bands). FGF-R1/PDGF-Rα coimmunoprecipitation was found in the absence (lane 2) and in the presence (lane 3) of FGF-2/PDGF-BB. Lane 4 reports PAE cells transfected with pcDNA3 empty vector, as control. They expressed very low levels of PDGF-Rα; thus, no signal was evident in Western blot for PDGF-Rα or for FGF-R1. Lane 1 represents the positive control for FGF-R1; it shows Western blot of cell lysates immunoprecipitated and immunoblotted with anti–FGF-R1 antibody and allows identification of FGF-R1 as the band observed in lanes 2 and 3. PDGF R-α was not detected in lane 1 as opposed to lanes 2 and 3, likely for a different steric impediment of the antibody couple used in lane 1 compared with the antibody couple used in lanes 2 and 3. (D) Lysates of untransfected HUVECs were immunoprecipitated with anti–PDGF-Rα antibodies and were analyzed by immunoblotting, probed with anti–FGF-R1 antibody (bottom bands), and reprobed with anti–PDGF-Rα antibodies (middle bands). FGF-R1/PDGF-Rα complex was found in untreated (lane 1) and FGF-2/PDGF-BB–treated (lane 2) cells. Immunoblotting with antiphosphotyrosine antibody (top bands) shows the activation of PDGF-Rα in lane 2. Lane 3 shows expression levels of FGF-R1 and PDGF-Rα by Western blot analysis of a cellular HUVEC lysate.

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