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Fig. 8. β-catenin activates the Vegfa promoter. 2.0-kb Vegfa promoter fragment was cloned into pGL4 vector and deletion mutants were generated. (A) RCJ3.1C5.18 chondrogenic cells were co-transfected with constitutively active β-catenin expression plasmid and the Vegfa promoter. Cell lysates were extracted 48 hours after transfection and luciferase was activity measured. β-catenin significantly stimulated VEGF-a promoter activity. (B) RCJ3.1C5.18 chondrocytes were transfected with the deletion constructs of the Vegfa promoter and the β-catenin expression plasmid. Luciferase activity was measured 48 hours after transfection. β-catenin activates Vegfa promoter activity in 2.0-kb, 1.34-kb and 0.94-kb fragments but not in the 0.14-kb fragment. The results suggest that the β-catenin-responsive region is located in the –940 to –140 region of the Vegfa promoter. *P<0.05, unpaired t-test, n=3 (A,B). (C) The TCF/LEF sites (TREs) in the Vegfa promoter are shown. (D) Chromatin IP assay was performed using RCJ3.1C5.18 cells treated with or without BIO (1 µg/ml) for 2 hours. IP was performed using anti-β-catenin antibody, and anti-Flag antibody was used as a negative control. The DNA-protein complex was crosslinked and used as PCR template. The ChIP assay showed that β-catenin binds to the Vegfa promoter and that addition of BIO to the cultures enhanced β-catenin binding.
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