Figure 5

Figure 5

VBP1 negatively modulates HIF-1α protein levels in a pVHL-independent manner.A, Overexpression of VBP1 decreased the HIF-1α protein levels in pVHL-deficient RCC4 cells. RCC4 cells were stably transfected with the control or pCS2-VBP1 plasmid, and the proteins were detected by Western blotting analysis. B, Protein levels of HIF-1α were elevated in VBP1 knockdown RCC4 cell lines. Immunoblot analysis of cells stably transfected with lentiviral pLKO plasmid with VBP1-specific shRNA or scrambled control shRNA. C, VBP1 decreases HIF-1α (P402A/P564A) protein levels. HEK293T/17 and HCT116 cells were transfected with the indicated plasmids, and the proteins were detected by Western blotting analysis. D, HIF luciferase reporter assay results in HeLa cells show that VBP1 decreased HIF-1α (P402A/P564A) transcriptional activity. Data are represented as means ± SD (n > 3 biological replicate samples). E, VBP1 interacts with HIF-1α (P402A/P564A). Co-IP was performed with lysates from HEK293T/17 cells co-transfected with plasmids overexpressing VBP1-Flag and HA-tagged HIF-1α (P402A/P564A). F, VBP1 has no effect on the protein levels of HIF-2α (P405A/P531A). Immunoblot analysis of cells transfected with the indicated plasmids. G, HIF luciferase reporter assay results in HEK293T/17 cells show that VBP1 does not affect the HIF-2α (P405A/P531A) transcriptional activity. ns, not significant. n > 3 biological replicate samples. H, Vbp1 negatively modulates the HIF-1α signaling pathway in Tg (hre-sv40mp:GFP) zebrafish in the vhl^−/− background. Tg(hre-sv40mp:GFP) embryos were injected with control mRNA or vbp1 mRNA, and images were taken in 72 hpf. The histograms show the average values (means ± SD) of GFP intensity in the somite of the Tg(hre-sv40mp:GFP) vhl^−/− larvae at 72 hpf. n/n, number of embryos showing representative GFP fluorenes/total number of embryos examined. I, Vbp1 negatively modulates the Hif-1α protein levels in vhl^−/− zebrafish. Zebrafish embryos injected with control mRNA or vbp1 mRNA were collected at 72 hpf and the proteins were detected by Western blotting analysis. HIF-1, Hypoxia-inducible factor-1; pVHL, von Hippel-Lindau protein; VBP1, pVHL binding protein 1.