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Fig. 3. (A) PEDF increases the kinase activity of Fes in IBE cells. IBE cells expressing either FLAG-tagged wild-type (WT6-8 cells) or FLAG-tagged kinase-inactive (KE5-15 cells) Fes were suspended in Ham's F-12 medium containing 0.25% BSA and seeded onto Matrigel. Cells were cultured with or without 200 ng ml–1 PEDF for 2 hours and lysed, and FLAG-tagged Fes was immunoprecipitated from 90% of cell lysate, followed by an in-vitro kinase assay. Relative activity was estimated using an Image Analyzer BAS 5000 (Fuji). The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. Reproducible results were obtained from two independent experiments. (B) PEDF inhibits FGF-2-induced activation of Fyn in IBE cells but not in KE5-15 cells. Cells were cultured on Matrigel in the presence or absence of the indicated samples for 2 hours. Cells were washed and lysed, and Fyn was immunoprecipitated from 90% of cell lysate, followed by an in-vitro kinase assay. Relative activity was estimated using an Image Analyzer BAS 5000. The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. Reproducible results were obtained from two independent experiments. (C) PEDF inhibits Fyn activity in HUVECs. HUVECs were suspended in EBM-2 medium containing 0.1% BSA and seeded onto Matrigel in the presence or absence of the indicated samples. Fyn was immunoprecipitated from 90% of cell lysate and an in-vitro kinase assay was performed. The remaining 10% of each lysate was assessed for the loaded amount of protein by immunoblotting. (D) Anti-pY529-Src antibody recognizes the C-terminal tyrosine phosphorylated Fyn. Src immunoprecipitated from HUVECs or Fyn precipitated from IBE cells was incubated with or without recombinant CSK and proteins were separated by SDS-PAGE, followed by transfer onto PVDF membranes. Phosphorylation of inhibitory tyrosine residue at the C-terminus was examined by immunoblotting with anti-pY529 Src antibody. (E) PEDF phosphorylates the inhibitory tyrosine residue at the C-terminus of Fyn in IBE cells but not in KE5-15 cells. Cells were cultured on Matrigel in the presence or absence of 200 ng ml–1 PEDF for 2 hours. Cells were washed and lysed, and Fyn was immunoprecipitated. Tyrosine phosphorylation of Fyn was estimated by immunoblotting. Reproducible results were obtained from two independent experiments. (F) Wild-type Fes, but not kinase-inactive Fes, phosphorylates the C-terminal tyrosine residue of Fyn. FLAG-tagged Fes was immunoprecipitated from either WT6-8 cells or KE5-15 cells cultured on Matrigel in the presence or absence of 200 ng ml–1 PEDF and incubated with recombinant human Fyn (rFyn) in the presence of ATP. As a positive control, rFyn was incubated with CSK. Phosphorylation of terminal tyrosine residue of rFyn was examined by immunoblotting.
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