Fig. 5.

HIF-2α and its target genes was regulated by functional mutant pVHL. a HA tagged wild type and mutant VHL (V155A, L158Q, Q164R) were transfected into 786-O cells. At 48 h post-transfection, cells were lysed in RIPA buffer and then the proteins which are related in UCP-VHL-HIF pathway and cyclin D1 were detected by immunoblot as indicated. b HeLa expressing shVHL was transfected with HRE-Luc plasmid and/or missense mutant pVHL expressing plasmid. At 48 h post-transfection, cells were lysed in luminol assay buffer. Functionality of missense mutant pVHL was revealed by luciferase activity. mRNA was purified from missense mutant VHL expressing 786-O stable cell line at 48 h after seeding and it was used for quantitation of transcripts of Glut-1(c) (n = 3, p < 0.01) and VEGF (d) (n = 3, p < 0.05) using LightCycler. e Cell proliferative changes of each mutant pVHL expressing 786-O stable cell lines were observed by following 1, 2, 3 days using hemocytometric counting method. f The number and the conditions of cell at final days was observed by hemocytometric counting (n = 3, p < 0.05) and crystal violet staining